Registration Dossier
Registration Dossier
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EC number: 287-722-1 | CAS number: 85567-10-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
To assess the genetic toxicity of the test item, three key in vitro genotoxicity assays were performed: (1) an Ames test, (2) a micronucleus assay, and (3) a mouse lymphoma assay. All tests were performed according to their specific OECD TG and GLP.
Ames assay (OECD 471)
The potential bacterial mutagenicity of the registered substance was tested up to 5000 µg/plate in a plate incorporation test with/without rat liver S9 (experiment I) and in the pre-incubation test with/without hamster liver S9 (experiment II) with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.
In vitro micronucleus assay (OECD 487)
The potential for induction of micronuclei in human lymphocytes was tested with the registered substance in an in vitro micronucleus assay at concentrations up to 2000 μg/mL with (4h exposure) and without metabolic activation (4h and 44h exposure).
No precipitate of the test item was noted in all concentration groups. In experiment I (4h exposure) without and with metabolic activation and in experiment II (44 h exposure) without metabolic activation no statistical or biologically relevant increase of the micronucleus frequency was noted after treatment with the test item.
The test item is concluded to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test.
In vitro mouse lymphoma assay (OECD 490)
The potential to induce mutations at the mouse lymphoma thymidine kinase locus was assessed using the cell line L5178Y. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay at the following concentrations: 100, 250, 500, 750, 1000, 1500 and 2000 µg/mL. The mutant frequencies induced by the Reactive Yellow 42 did not show any biologically relevant increase. In conclusion, Reactive Yellow 42 is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
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:
- 2016-2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial
Reverse Mutation Test", adopted 21st July, 1997. - Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- May 30, 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- EPA 712-C-98-247, August 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Rat liver (Experiment I), S9 Hamster Liver (Experiment II)
- Test concentrations with justification for top dose:
- Pre-Experiment for Toxicity:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment I:
31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment II:
10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
The test item concentrations for main experiments were chosen based on pre-experiment.
5000 μg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Aqua dest.
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity. - Untreated negative controls:
- yes
- Remarks:
- negative control = solvent control Aqua dest.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- negative control = solvent control Aqua dest.
- Positive controls:
- yes
- Remarks:
- S. typhimurium: TA 100, TA 1535 without metabolic activation dissolved in Aqua dest.
- Positive control substance:
- sodium azide
- Positive controls:
- yes
- Remarks:
- S. typhimurium: TA 98, TA 1537 without metabolic activation dissolved in DMSO
- Positive control substance:
- other: 4-nitro-o-phenylene-diamine (4-NOPD)
- Positive controls:
- yes
- Remarks:
- S. typhimurium: TA 102 without metabolic activation dissolved in Aqua dest.
- Positive control substance:
- methylmethanesulfonate
- Positive controls:
- yes
- Remarks:
- S. typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 102 with metabolic activation (rat liver) dissolved in DMSO
- Positive control substance:
- other: 2-aminoanthracene (2-AA)
- Positive controls:
- yes
- Remarks:
- S. typhimurium: TA 1535, TA 1537, TA 102 with metabolic activation (hamster liver) dissolved in DMSO
- Positive control substance:
- other: 2-aminoanthracene (2-AA)
- Positive controls:
- yes
- Remarks:
- S. typhimurium: TA 98, TA 100 with metabolic activation (hamster liver) dissolved in Aqua dest.
- Positive control substance:
- congo red
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Experiment I: in medium; in agar (plate incorporation)
Experiment II: preincubation
DURATION
Experiment I :
- Preincubation period: No
- Exposure duration: at least 48 h in the dark at 37°C
- Selection time (if incubation with a selection agent): at least 48 h in the dark at 37°C
Experiment II:
- Preincubation period: 30 min at 30°C
- Exposure duration: 30 min at 30°C and at least 48 h in the dark at 37°C
- Selection time (if incubation with a selection agent): at least 48 h in the dark at 37°C
SELECTION AGENT (mutation assays): histidine
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: other: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control - Evaluation criteria:
- The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system. - Statistics:
- According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- Experiment I and II
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- Experiment I and II
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Experiment I: toxic effects of the test item at a concentration of 5000 μg/plate (with and without metabolic activation).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Experiment II: toxic effects of the test item at concentrations of 2500 μg/plate and higher (with metabolic activation).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Remarks:
- Experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a pre-experiment.
Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test).
The test item was tested in the pre-experiment with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
No toxicity of the test item was noted in the pre-experiment with tester strains TA 98 and TA 100
COMPARISON WITH HISTORICAL CONTROL DATA:
The negative control plates (Aqua dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range
(2013 -2015)):
-S9 + S9 (rat liver)
min max min max
TA 98 13 54 13 61
TA 100 49 139 67 162
TA 1535 4 39 4 32
TA 1537 2 35 3 36
TA 102 141 472 91 586
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects of the test item were noted in some tester strains used in experiment I and II:
- In experiment I toxic effects of the test item were observed in tester strain TA 1535 at 5000 μg/plate (with and without metabolic activation).
- In experiment II toxic effects of the test item were noted in tester strain TA 1537 at 2500 μg/plate and higher (with metabolic activation).
The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in experiment I in tester strain TA 1535 and 1537 at a concentration of 316 μg/plate and 100 μg/plate (with metabolic activation), respectively was regarded as not biologically relevant due to lack of a dose-response relationship. - Conclusions:
- Under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.
- Executive summary:
In order to investigate the potential of the test item for its ability to induce gene mutations the plate incorporation test with rat liver S9 (experiment I) and the pre-incubation test with hamster liver S9 (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:
Experiment I: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment II: 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
Toxic effects of the test item were noted in some tester strains used in experiment I and II:
- In experiment I toxic effects of the test item were observed in tester strain TA 1535 at a concentration of 5000 μg/plate (with and without metabolic activation).
- In experiment II toxic effects of the test item were noted in tester strain TA 1537 at concentrations of 2500 μg/plate and higher (with metabolic activation).
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Reactive Yellow 42 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
All criteria of validity were met.
In conclusion, under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- - Principle of test: in vitro test to assess the potential of the test item to induce gene mutations by means of a Thymidine Kinase assay using the mouse lymphoma cell line L5178Y. The Thymidine Kinase (TK) system detects base pair mutations, frameshift mutations, small deletions as well as large, non-lethal deletions and rearrangements of the relevant chromosomes.
- Short description of test conditions: the experiment with and without metabolic activation was performed as 4 hours short-term exposure assay
- Parameters analysed / observed: potential mutagenicity and/or clastogencity - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: In vitro Mammalian Cell Gene Mutation Assay in Mouse Lymphoma L5178Y Cells
- Target gene:
- Thymidine Kinase Locus/TK+/-)
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Eurofins Munich stock cultures
- Suitability of cells: successfully in in vitro experiments for many years.
- Cell cycle length, doubling time or proliferation index: 10-12 h doubling time
- Methods for maintenance in cell culture if applicable: Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase) allowing the repeated use of the same cell batch in experiments. Each cell batch is routinely checked for mycoplasma infection.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 supplemented with hypoxanthine, thymidine, glycine, ethotrexate. The cells are resuspended in medium without methotrexate but thymidine, hypoxanthine and glycine for 1-3 days. - Metabolic activation:
- with and without
- Metabolic activation system:
- Mammalian Microsomal Fraction S9 Homogenate
- Test concentrations with justification for top dose:
- Concentrations: 100, 250, 500, 750, 1000, 1500, 2000 µg/mL.
Based on results (and absence of toxicity) observed in the micronucleus assay, 2000 µg/mL was selected as the highest dose. - Vehicle / solvent:
- A solublity test was performed with different solvent and vehicle up to a maximum concentration of 5 mg/mL. Based on the results of the solubility test RPMI cell culture medium was used as solvent (RPMI + 5% HS).
The stock solutions were prepared directly prior to treatment. The solvent was compatible with the survival of the cells and the S9 activities. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- ethylmethanesulphonate
- other: methylmethanesulfonate (MMS)
- Remarks:
- The dilutions of the stock solutions of the positive controls were prepared on the day of the experiment and used immediately.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; 11 mL RPMI medium with 5% horse serum (25 cm2 flasks)
- Cell density at seeding (if applicable): 1 x 10E7 cells
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Density check: each days and adjusted to 3x10E5 cells/mL. (total volume 20mL)
NUMBER OF REPLICATIONS: 4
NUMBER OF CELLS EVALUATED: the plates were scored after incubation period of 12 days
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RSG) - Evaluation criteria:
- The test item is considered mutagenic if the following criteria are met:
- the induced mutant frequency meets or exceeds the Global Evaluation Factor (GEF) of 126 mutants per 10E6 cells and
- a dose-dependent increase in mutant frequency is detected
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥ 40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal abberations.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative. - Statistics:
- According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the results.
For the microwell method used here the Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection.
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- 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
- Additional information on results:
- Please see the table under the section "Any other information on results".
The acceptance criteria as recommended by IWGT were fulfilled - Conclusions:
- The test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay in mouse lymphoma L5178Y cells.
- Executive summary:
In this in vitro study Mammalian Cell Gene mutation Assay, the mutagen potential of the test item Reactive Yellow 42 was assessed according to the OECD 490 and under GLP without significant deviations.
The test item Reactive Yellow 42 was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The mutant selection cloning was performed by using liquid medium in 96 -microwell plates. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. The test item was investigated at the following concentrations (with and without metabolic activation): 100, 250, 500, 750, 1000, 1500 and 2000 µg/mL.
No precipitation of the test item was observed.
No growth precipitation was observed in the experiment with and without metabolic activation. The relative total growth (RTG) of 45.2% at a concentration of 1500 µg/mL was regarded as not biologically relevant due to lack of dose-response relationship.
The mutant frequency obtained from all experiments were compared with the Global Evaluation Factor (GEF) and a statistical analysis was performed.
The positive controls EMS, MMS and B(a)P showed distinct effects in mutation frequency, thus, providing the validity of the test system to detect potential mutagenic effects.
In the main experiment with and without metabolic activation, all validity criteria were met. The negative controls showed mutant frequencies within the acceptance range of 50 -170 mutants/106 cells, according to the IWTG criteria.
The mutant frequencies induced by the Reactive Yellow 42 did not show any biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups. A statistical analysis displayed that with metabolic activation some of the mutant frequencies were significantly increased over those of the negative controls. As the corresponding GEF was not exceeded, this effect was considered as not biologically relevant.
The colony sizing was performed for the highest concentration of the test item and for the negative and positive controls.
An extension of the GEF by the induced mutant frequency in combination with an increased occurrence of small colonies is an indication of potential clastogenic effects and/or chromosal abberations. The positive controls MMS and B(a)P induced a significant increase in mutant frequency and a biologically significant increase of small colonies (≥ 40%), thus proving the ability of the test system to indicate potential clastogenic effects. In the main experiment with and without metabolic activation the percentage of small colonies in the negative controls and in the highest dose groups treated with the test item was found to be lower than 40%. Thus, all dose groups were considered as not clastogenic.
In the main experiment with and without metabolic activation all validity criteria were met.
In conclusion, nder the experimental conditions reported, Reactive Yellow 42 is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- adopted 29 July, 2016
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes: human
- Remarks:
- from a single donor (male, younger than 35 years)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Human peripheral blood lymphocytes from healthy and non-smoking donors, - Suitability of cells:
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable: single donor, male, younger than 35 years
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood samples treated with an anti-coagulant (e. g. heparin) were pre-cultured in the presence of mitogen (phytohaematogglutinin, PHA).
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Complete Culture Medium
RPMI 1640 medium supplemented with:
- 15 % fetal bovine serum (FBS)
- 100 U/100 μg/mL penicillin/streptomycin solution
- 2.4 μg/mL phytohaemagglutinin (PHA)
Treatment Medium (short-term exposure): Complete culture medium without FBS.
After Treatment Medium / Treatment Medium (long-term exposure): Complete culture medium with 15 % FBS and 6 μg/mL cytochalasin B. - Cytokinesis block (if used):
- 6 μg/mL cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix rat liver
- Test concentrations with justification for top dose:
- -Pre-experiment: without and with S9 mix: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 1500 and 2000 μg/mL
-Experiment I with short-term exposure (4 h): without and with metabolic activation: 125, 250, 500, 1000, 1500 and 2000 μg/mL
-Experiment II with long-term exposure (44 h): without metabolic activation: 50, 100, 250, 500, 1000, 1500 and 2000 μg/mL
-main experiment for the microscopic analyses:
Experiment I with short-term exposure (4 h):
without metabolic activation: 1000, 1500 and 2000 μg/mL
with metabolic activation: 500, 1500 and 2000 μg/mL
Experiment II with long-term exposure (44 h):
without metabolic activation: 1000, 1500 and 2000 μg/mL
The concentration of 2000 μg/mL was considered to be the highest test concentration to be used in this test system following the recommendation of the corresponding OECD testing guideline 487.
The selection of the concentrations used in experiment I and II based on data from the pre-experiment. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: cell culture medium (RPMI: Roswell Park Memorial Institute medium)
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity. - Untreated negative controls:
- yes
- Remarks:
- cell culture medium
- Positive controls:
- yes
- Remarks:
- without metabolic activation, dissolved in RPMI
- Positive control substance:
- ethylmethanesulphonate
- Positive controls:
- yes
- Remarks:
- with metabolic activation, dissoved in RPMI
- Positive control substance:
- cyclophosphamide
- Positive controls:
- yes
- Remarks:
- without metabolic activation, dissolved in RPMI
- Positive control substance:
- other: colchicine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
Experiment I (4 h exposure)
- Exposure duration: 4 h
- Expression time (cells in growth medium): 40 h to 42 h at 37°C and 5% CO2
- Fixation time (start of exposure up to fixation or harvest of cells): 44 h
- Without and with S9
DURATION
Experiment II
- Exposure duration: 44 h
- Expression time (cells in growth medium): 44 h
- Fixation time (start of exposure up to fixation or harvest of cells): 44 h
- Without S9
SPINDLE INHIBITOR (cytogenetic assays): 6 μg/mL cytochalasin B
STAIN (for cytogenetic assays): acridine orangesolution (10 µg/mL)
NUMBER OF REPLICATIONS: Duplicate cultures were performed at each concentration level except for the pre-experiment.
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
At the end of the cultivation, the complete culture medium was removed. Subsequently, the cells were treated with cold hypotonic solution (0.075 M KCl) for some minutes at room temperature and immediately centrifuged. The pellet was resuspended with a solution consisted of fixation solution + NaCl 0.9% (1+1) and centrifuged. After that the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides.
Consecutively, the cells were dried on a heating plate. The cells were stained with acridine orange by incubating the slides in acridine orange solution (10 μg/mL) for one minute, a consecutive washing step with phosphate buffered saline and distilled water and letting them air-dry.
NUMBER OF CELLS EVALUATED:
For each dose group at least 2000 binucleated cells (if possible) per concentration (1000 binucleated cells per slide) were analysed for micronuclei.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
according to the criteria of Fenech, i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered
DETERMINATION OF CYTOTOXICITY
- Method: other: The CBPI (cytokinesis block proliferation index) was used for the quantification of cytotoxicity in the Pre-experiment for toxicity.
As an assessment of the cytotoxicity, a cytokinesis block proliferation index (CBPI) was determined from 500 cells according to the following formula:
CBPI=(c1 x 1) + (c2 x 2) + (cx x 3)
n
c1: mononucleate cells
c2: binucleate cells
cx: multinucleate cells
n: total number of cells
The CBPI was used to calculate the % cytostasis, which indicates the inhibition of cell growth of treated cultures in comparison to control cultures:
% Cytostasis= 100 – 100 x ((CBPIT – 1) / (CBPIC – 1))
CBPIT: Cytokinesis Block proliferation index of treated cultures
CBPIC: Cytokinesis Block proliferation index of control cultures - Evaluation criteria:
- The assay is considered acceptable if it meets the following criteria:
- Concurrent negative control = acceptable for addition to the laboratory historical negative control database.
- Concurrent positive controls should induce responses compatible with those generated in the labs historical positive control database & produce a statistically significant increase compared with the concurrent negative control.
- Cell proliferation criteria in the negative control should be fulfilled.
- All experimental conditions are tested unless one resulted in positive results.
- Adequate number of cells and concentrations are analysable.
- Criteria for the selection of top concentration are fulfilled.
A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met. - Statistics:
- The nonparametric χ² Test was performed to verify the results in Experiments I and II.
The χ ² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. - Key result
- Species / strain:
- lymphocytes: human peripheral blood
- Remarks:
- Experiment I (4 h exposure)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: no increase of the cytostasis above 30% was noted.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: human peripheral blood
- Remarks:
- Experiment II (44 h exposure)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: at a concentration of 1500 μg/mL a cytostasis of 31% was noted but was considered as not biologically relevant due to lack of a dose-response relationship
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH test item within physiological range.
- Effects of osmolality: For the maximum concentration without metabolic activation the osmolality (in comparison to negative (solvent) control) using an OSMOMAT 3000 (Fa. Gonotec) and pH value using pH test paper were determined:
Concentration (µg/mL) Osmolality (mOsm/kg) pH value
Exp. I Solvent control - 290 -
Test item 2000 309 6.6
Exp. II Solvent control - 313 -
Test item 2000 296 6.6
- Precipitation: no precipitate of test item in any group in main test
RANGE-FINDING/SCREENING STUDIES:
- As per OECD guideline: highest recommended concentration = 2000 μg/mL. The test item was dissolved in cell culture medium & no precipitate was noted.
- highest dose group pre-experiment = 2000 μg/mL
- cytokinesis block proliferation index (CBPI) was used to calculate the cytostasis (cytostasis [%] = 100 - CBPI relative [%]). The cytostasis was used to describe cytotoxicity.
The selection of concentrations used in the main experiment based on the results obtained in the pre-experiment.
COMPARISON WITH HISTORICAL CONTROL DATA:
historical control limits of the negative control micronucleated cell frequency in:
- experiment I without metabolic activation : 0.35% – 1.33%
- experiment I with metabolic activation: 0.32% – 1.53%
- experiment II without metabolic activation : 0.35% – 1.33%
ADDITIONAL INFORMATION ON CYTOTOXICITY:
If cytotoxicity is observed the highest concentration evaluated should not exceed the limit of 55% ± 5% cytotoxicity according to the OECD 487. Higher levels of cytotoxicity may induce chromosome damage as a secondary effect of cytotoxicity. The other concentrations evaluated should exhibit intermediate and little or no toxicity. OECD 487 does not define the limit for discriminating between cytotoxic and non-cytotoxic effects. According to laboratory experience this limit is a value of the relative cell growth of 70% compared to the negative control which corresponds to 30% of cytostasis. In experiment I with and without metabolic activation no increase of the cytostasis above 30% was noted. In experiment II without metabolic activation at a concentration of 1500 μg/mL a cytostasis of 31% was noted but was considered as not biologically relevant due to lack of a dose-response relationship. - Conclusions:
- Under the reported experimental conditions, the test item did not induce structural and/or numerical chromosomal damage in human lymphocytes. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test.
- Executive summary:
In order to investigate a possible potential of the test item for its ability to induce micronuclei in human lymphocytes an in vitro micronucleus assay was carried out. The following study design was performed:
Without S9
With S9
Exp. I
Exp. II
Exp. I
Exposure period
4 h
44 h
4 h
Cytochalasin B exposure
40 h
43 h
40 h
Preparation interval
44 h
44 h
44 h
Total culture period*
92 h
92 h
92 h
*Exposure started 48 h after culture initiation
The selection of the concentrations was based on data from the pre-experiment. In the main experiment without and with metabolic activation 2000 μg/mL test item was selected as the highest concentration.
The following concentrations were evaluated for micronuclei frequencies:
- Experiment I with short-term exposure (4 h): without metabolic activation: 1000, 1500 and 2000 μg/mL and with metabolic activation: 500, 1500 and 2000 μg/mL
- Experiment II with long-term exposure (44 h): without metabolic activation: 1000, 1500 and 2000 μg/mL
No precipitate of the test item was noted in any concentration group evaluated in the main experiment.
In experiment I with and without metabolic activation no increase of the cytostasis above 30 % was noted. In experiment II without metabolic activation at a concentration of 1500 μg/mL a cytostasis of 31% was noted but was considered as not biologically relevant due to lack of a dose-response relationship.
In experiment I without and with metabolic activation and in experiment II without metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item.
The nonparametric χ² Test was performed to verify the results in both experiments. No statistically significant enhancement (p<0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment I and II. The χ ² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II.
Ethylmethanesulfonate (EMS, 600 μg/mL and 1400 μg/mL) and cyclophosphamide (CPA, 15 μg/mL) were used as clastogenic controls. Colchicine (Colc, 0.02 μg/mL and 0.8 μg/mL) was used as aneugenic control. All induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in human lymphocytes. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test.
Referenceopen allclose all
Table 1: Results Pre-Experiment
Substance |
Dose (μg/plate) |
TA 98 Mutation Factor [toxicity]* |
TA 100 Mutation Factor [toxicity]* |
||
without S9 |
with S9 |
without S9 |
with S9 |
||
Solvent Control (A. dest) |
|
1.0 |
1.0 |
1.0 |
1.0 |
4-NOPD |
10.0 |
19.5 |
- |
- |
- |
NaN3 |
10.0 |
- |
- |
6.1 |
- |
2-AA |
2.50 |
- |
76.0 |
- |
13.2 |
Test Item
|
3.16 |
1.3 |
1.0 |
1.0 |
0.9 |
10.0 |
1.2 |
0.9 |
1.1 |
1.1 |
|
31.6 |
1.3 |
0.9 |
1.0 |
1.0 |
|
100 |
1.1 |
1.0 |
1.0 |
1.0 |
|
316 |
1.4 |
0.7 |
1.1 |
0.9 |
|
1000 |
1.4 |
0.8 |
1.1 |
0.9 |
|
2500 |
1.4 |
0..9 |
1.1 |
1.0 |
|
5000 |
1.1 |
0.9 |
1.2 |
0.9 |
|
* [toxicity parameter]: B = Background lawn reduced; N = No background lawn
Table 2: Results Experiment I (Plate-incorporation Test; Rat S9)
Tester Strain: TA 98 Experiment 1
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||
Without activation (-S9) |
With activation (+S9) |
||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||
A. dest |
|
18 |
18 |
2.5 |
34 |
34 |
1.5 |
1.0 |
1.0 |
16 |
35 |
||||||||
21 |
32 |
||||||||
Test item |
31.6 µg |
17 |
23 |
5.7 |
31 |
30 |
5.0 |
1.3 |
0.9 |
25 |
35 |
||||||||
28 |
25 |
||||||||
Test item |
100 µg |
21 |
21 |
4.0 |
40 |
35 |
7.0 |
1.1 |
1.0 |
17 |
38 |
||||||||
25 |
27 |
||||||||
Test item |
316 µg |
18 |
26 |
10.0 |
28 |
24 |
3.8 |
1.4 |
0.7 |
22 |
21 |
||||||||
37 |
22 |
||||||||
Test item |
1000 µg |
16 |
25 |
8.1 |
23 |
27 |
4.0 |
1.4 |
0.8 |
31 |
31 |
||||||||
29 |
27 |
||||||||
Test item |
2500 µg |
28 |
25 |
3.8 |
33 |
31 |
13.1 |
1.4 |
0.9 |
21 |
43 |
||||||||
27 |
17 |
||||||||
Test item |
5000 µg |
17 |
19 |
4.0 |
28 |
30 |
4.9 |
1.1 |
0.9 |
24 |
36 |
||||||||
17 |
27 |
||||||||
4-NOPD |
10 µg |
305 |
358 |
109.2 |
/ |
/ |
/ |
19.5 |
/ |
484 |
/ |
||||||||
286 |
/ |
||||||||
2-AA |
2.5 µg |
/ |
/ |
/ |
2067 |
2560 |
427.1 |
/ |
76.0 |
/ |
2816 |
||||||||
/ |
2797 |
||||||||
SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn
Mutation factor = mean revertants (test item)
mean revertants (vehicle control)
Tester Strain: TA 100 Experiment 1
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||
Without activation (-S9) |
With activation (+S9) |
||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||
A. dest |
|
148 |
136 |
14.3 |
128 |
129 |
1.0 |
1.0 |
1.0 |
120 |
129 |
||||||||
139 |
130 |
||||||||
Test item |
31.6 µg |
117 |
141 |
21.9 |
125 |
126 |
3.2 |
1.0 |
1.0 |
146 |
124 |
||||||||
160 |
130 |
||||||||
Test item |
100 µg |
154 |
137 |
15.0 |
123 |
131 |
9.8 |
1.0 |
1.0 |
133 |
128 |
||||||||
125 |
142 |
||||||||
Test item |
316 µg |
136 |
143 |
14.5 |
118 |
120 |
15.1 |
1.1 |
0.9 |
160 |
106 |
||||||||
134 |
136 |
||||||||
Test item |
1000 µg |
167 |
150 |
15.4 |
105 |
116 |
11.0 |
1.1 |
0.9 |
146 |
127 |
||||||||
137 |
116 |
||||||||
Test item |
2500 µg |
151 |
154 |
3.6 |
144 |
132 |
12.6 |
1.1 |
1.0 |
153 |
119 |
||||||||
158 |
134 |
||||||||
Test item |
5000 µg |
166 |
158 |
13.0 |
116 |
119 |
2.6 |
1.2 |
0.9 |
143 |
120 |
||||||||
165 |
121 |
||||||||
NaN3 |
10 µg |
770 |
833 |
62.0 |
/ |
/ |
/ |
6.1 |
/ |
894 |
/ |
||||||||
834 |
/ |
||||||||
2-AA |
2.5 µg |
/ |
/ |
/ |
1539 |
1703 |
216.0 |
/ |
13.2 |
/ |
1623 |
||||||||
/ |
1948 |
||||||||
SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn
Mutation factor = mean revertants (test item)
mean revertants (vehicle control)
Tester Strain: TA 1535 Experiment 1
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||
Without activation (-S9) |
With activation (+S9) |
||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||
A. dest |
|
11 |
14 |
2.5 |
16 |
14 |
4.0 |
1.0 |
1.0 |
14 |
16 |
||||||||
16 |
9 |
||||||||
Test item |
31.6 µg |
11 |
14 |
8.5 |
6 |
8 |
5.7 |
1.0 |
0.6 |
8 |
14 |
||||||||
24 |
3 |
||||||||
Test item |
100 µg |
11 |
10 |
0.6 |
13 |
10 |
3.6 |
0.8 |
0.7 |
10 |
11 |
||||||||
10 |
6 |
||||||||
Test item |
316 µg |
7 |
10 |
3.5 |
11 |
6 |
4.2 |
0.7 |
0.5 |
12 |
3 |
||||||||
/ |
5 |
||||||||
Test item |
1000 µg |
13 |
10 |
4.4 |
8 |
11 |
2.5 |
0.7 |
0.8 |
12 |
13 |
||||||||
5 |
11 |
||||||||
Test item |
2500 µg |
16 |
13 |
6.4 |
7 |
8 |
1.5 |
1.0 |
0.6 |
18 |
10 |
||||||||
6 |
8 |
||||||||
Test item |
5000 µg |
6 |
7 |
1.7 |
6 |
6 |
0.6 |
0.5 |
0.5 |
9 |
6 |
||||||||
6 |
7 |
||||||||
NaN3 |
10 µg |
929 |
947 |
82.9 |
/ |
/ |
/ |
69.3 |
/ |
874 |
/ |
||||||||
1037 |
/ |
||||||||
2-AA |
2.5 µg |
/ |
/ |
/ |
196 |
179 |
16.5 |
/ |
13.1 |
/ |
163 |
||||||||
/ |
179 |
||||||||
SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn
Mutation factor = mean revertants (test item)
mean revertants (vehicle control)
Tester Strain: TA 1537 Experiment 1
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||
Without activation (-S9) |
With activation (+S9) |
||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||
A. dest |
|
10 |
10 |
3.0 |
12 |
9 |
2.3 |
1.0 |
1.0 |
7 |
8 |
||||||||
13 |
8 |
||||||||
Test item |
31.6 µg |
14 |
12 |
2.1 |
2 |
6 |
3.8 |
1.2 |
0.7 |
10 |
8 |
||||||||
11 |
9 |
||||||||
Test item |
100 µg |
12 |
12 |
2.5 |
6 |
4 |
1.5 |
1.2 |
0.5 |
9 |
3 |
||||||||
14 |
4 |
||||||||
Test item |
316 µg |
11 |
10 |
0.6 |
6 |
7 |
2.6 |
1.0 |
0.8 |
10 |
5 |
||||||||
10 |
10 |
||||||||
Test item |
1000 µg |
11 |
11 |
0.0 |
7 |
6 |
2.3 |
1.1 |
0.6 |
11 |
7 |
||||||||
11 |
3 |
||||||||
Test item |
2500 µg |
11 |
16 |
4.5 |
7 |
6 |
2.1 |
1.6 |
0.7 |
20 |
8 |
||||||||
16 |
4 |
||||||||
Test item |
5000 µg |
17 |
12 |
5.0 |
12 |
11 |
4.0 |
1.2 |
1.2 |
11 |
7 |
||||||||
7 |
15 |
||||||||
4-NOPD |
40 µg |
98 |
104 |
8.5 |
/ |
/ |
/ |
10.4 |
/ |
114 |
/ |
||||||||
101 |
/ |
||||||||
2-AA |
2.5 µg |
/ |
/ |
/ |
313 |
352 |
31.3 |
/ |
37.7 |
/ |
388 |
||||||||
/ |
337 |
||||||||
SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn
Mutation factor = mean revertants (test item)
mean revertants (vehicle control)
Tester Strain: TA 102 Experiment 1
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||
Without activation (-S9) |
With activation (+S9) |
||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||
A. dest |
|
299 |
280 |
20.7 |
307 |
321 |
24.5 |
1.0 |
1.0 |
258 |
306 |
||||||||
283 |
349 |
||||||||
Test item |
31.6 µg |
257 |
256 |
34.0 |
398 |
356 |
41.5 |
0.9 |
1.1 |
290 |
355 |
||||||||
222 |
315 |
||||||||
Test item |
100 µg |
220 |
219 |
10.0 |
285 |
286 |
7.5 |
0.8 |
0.9 |
209 |
279 |
||||||||
229 |
294 |
||||||||
Test item |
316 µg |
214 |
220 |
4.9 |
261 |
258 |
6.7 |
0.8 |
0.8 |
223 |
262 |
||||||||
222 |
250 |
||||||||
Test item |
1000 µg |
202 |
242 |
42.8 |
217 |
234 |
44.6 |
0.9 |
0.7 |
236 |
201 |
||||||||
287 |
285 |
||||||||
Test item |
2500 µg |
255 |
251 |
6.7 |
288 |
252 |
32.6 |
0.9 |
0.8 |
243 |
225 |
||||||||
254 |
242 |
||||||||
Test item |
5000 µg |
271 |
271 |
16.0 |
283 |
261 |
32.8 |
1.0 |
0.9 |
255 |
223 |
||||||||
287 |
276 |
||||||||
MMS |
1 µL |
1478 |
1480 |
4.0 |
/ |
/ |
/ |
5.3 |
/ |
1478 |
/ |
||||||||
1485 |
/ |
||||||||
2-AA |
10 µg |
/ |
/ |
/ |
964 |
978 |
19.9 |
/ |
3.1 |
/ |
970 |
||||||||
/ |
1001 |
||||||||
SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn
Mutation factor = mean revertants (test item)
mean revertants (vehicle control)
Table 3: Results Experiment II (Pre-incubation Test; Hamster S9)
Tester Strain: TA 98 Experiment 2
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||
Without activation (-S9) |
With activation (+S9) |
||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||
A. dest |
|
19 |
21 |
1.7 |
50 |
43 |
9.1 |
1.0 |
1.0 |
22 |
47 |
||||||||
22 |
33 |
||||||||
Test item |
10.0 µg |
14 |
19 |
4.4 |
52 |
41 |
10.1 |
0.9 |
1.0 |
22 |
32 |
||||||||
21 |
40 |
||||||||
Test item |
31.6 µg |
23 |
19 |
4.4 |
50 |
47 |
10.4 |
0.9 |
1.1 |
15 |
35 |
||||||||
16 |
55 |
||||||||
Test item |
100 µg |
20 |
20 |
4.5 |
51 |
42 |
12.3 |
1.0 |
1.0 |
16 |
47 |
||||||||
25 |
28 |
||||||||
Test item |
316 µg |
11 |
17 |
5.3 |
41 |
39 |
2.6 |
0.8 |
0.9 |
21 |
36 |
||||||||
19 |
40 |
||||||||
Test item |
1000 µg |
19 |
19 |
1.5 |
50 |
50 |
8.0 |
0.9 |
1.2 |
21 |
58 |
||||||||
18 |
42 |
||||||||
Test item |
2500 µg |
21 |
19 |
5.3 |
33 |
30 |
2.5 |
0.9 |
0.7 |
23 |
28 |
||||||||
13 |
30 |
||||||||
Test item |
5000 µg |
19 |
18 |
4.0 |
28 |
32 |
6.1 |
0.9 |
0.7 |
14 |
39 |
||||||||
22 |
29 |
||||||||
Congo Red |
700 µg |
/ |
/ |
/ |
108 |
114 |
25.0 |
/ |
2.6 |
/ |
141 |
||||||||
/ |
92 |
||||||||
4-NODP |
10 µg |
343 |
374 |
41.4 |
/ |
/ |
/ |
17.8 |
/ |
421 |
/ |
||||||||
358 |
/ |
||||||||
SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn
Mutation factor = mean revertants (test item)
mean revertants (vehicle control)
Tester Strain: TA 100 Experiment 2
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||
Without activation (-S9) |
With activation (+S9) |
||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||
A. dest |
|
93 |
93 |
0.6 |
84 |
89 |
4.2 |
1.0 |
1.0 |
94 |
90 |
||||||||
93 |
92 |
||||||||
Test item |
10.0 µg |
100 |
93 |
8.2 |
87 |
95 |
10.8 |
1.0 |
1.1 |
84 |
90 |
||||||||
95 |
107 |
||||||||
Test item |
31.6 µg |
94 |
110 |
17.1 |
105 |
110 |
13.2 |
1.2 |
1.2 |
108 |
125 |
||||||||
128 |
100 |
||||||||
Test item |
100 µg |
126 |
116 |
10.0 |
117 |
111 |
7.9 |
1.2 |
1.3 |
115 |
102 |
||||||||
106 |
114 |
||||||||
Test item |
316 µg |
101 |
110 |
13.9 |
118 |
120 |
2.9 |
1.2 |
1.3 |
126 |
118 |
||||||||
103 |
123 |
||||||||
Test item |
1000 µg |
85 |
105 |
18.0 |
124 |
113 |
17.1 |
1.1 |
1.3 |
119 |
93 |
||||||||
112 |
121 |
||||||||
Test item |
2500 µg |
92 |
100 |
12.2 |
91 |
99 |
12.7 |
1.1 |
1.1 |
94 |
93 |
||||||||
114 |
114 |
||||||||
Test item |
5000 µg |
102 |
104 |
6.7 |
80 |
87 |
10.2 |
1.1 |
1.0 |
111 |
83 |
||||||||
98 |
99 |
||||||||
Congo Red |
700 µg |
/ |
/ |
/ |
244 |
262 |
16.3 |
/ |
3.0 |
/ |
268 |
||||||||
/ |
275 |
||||||||
NaN3 |
10 µg |
720 |
731 |
37.7 |
/ |
/ |
/ |
7.8 |
/ |
700 |
/ |
||||||||
773 |
/ |
||||||||
SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn
Mutation factor = mean revertants (test item)
mean revertants (vehicle control)
Tester Strain: TA 1535 Experiment 2
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||
Without activation (-S9) |
With activation (+S9) |
||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||
A. dest |
|
28 |
34 |
5.2 |
15 |
15 |
1.5 |
1.0 |
1.0 |
37 |
13 |
||||||||
37 |
16 |
||||||||
Test item |
10.0 µg |
18 |
19 |
2.3 |
14 |
12 |
1.7 |
0.6 |
0.8 |
18 |
11 |
||||||||
22 |
11 |
||||||||
Test item |
31.6 µg |
23 |
27 |
4.0 |
8 |
10 |
2.5 |
0.8 |
0.7 |
28 |
10 |
||||||||
31 |
13 |
||||||||
Test item |
100 µg |
26 |
21 |
4.2 |
13 |
11 |
2.5 |
0.6 |
0.7 |
20 |
8 |
||||||||
18 |
11 |
||||||||
Test item |
316 µg |
26 |
26 |
2.5 |
8 |
10 |
2.5 |
0.8 |
0.7 |
28 |
10 |
||||||||
23 |
13 |
||||||||
Test item |
1000 µg |
22 |
23 |
2.6 |
10 |
12 |
4.4 |
0.7 |
0.8 |
26 |
17 |
||||||||
21 |
9 |
||||||||
Test item |
2500 µg |
17 |
19 |
6.2 |
15 |
13 |
2.1 |
0.6 |
0.9 |
14 |
11 |
||||||||
26 |
12 |
||||||||
Test item |
5000 µg |
26 |
26 |
0.6 |
16 |
21 |
8.4 |
0.8 |
1.5 |
26 |
17 |
||||||||
27 |
31 |
||||||||
NaN3 |
10 µg |
944 |
942 |
2.6 |
/ |
/ |
/ |
27.7 |
/ |
939 |
/ |
||||||||
943 |
/ |
||||||||
2-AA |
2.5 µg |
/ |
/ |
/ |
200 |
217 |
16.5 |
/ |
14.8 |
/ |
218 |
||||||||
/ |
233 |
||||||||
SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn
Mutation factor = mean revertants (test item)
mean revertants (vehicle control)
Tester Strain: TA 1537 Experiment 2
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||
Without activation (-S9) |
With activation (+S9) |
||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||
A. dest |
|
11 |
7 |
3.5 |
14 |
22 |
10.8 |
1.0 |
1.0 |
7 |
34 |
||||||||
4 |
17 |
||||||||
Test item |
10.0 µg |
5 |
7 |
2.1 |
13 |
17 |
3.6 |
0.9 |
0.8 |
6 |
18 |
||||||||
9 |
20 |
||||||||
Test item |
31.6 µg |
9 |
11 |
3.2 |
16 |
17 |
1.2 |
1.5 |
0.8 |
10 |
18 |
||||||||
15 |
18 |
||||||||
Test item |
100 µg |
4 |
5 |
1.5 |
14 |
15 |
1.5 |
0.7 |
0.7 |
5 |
15 |
||||||||
7 |
17 |
||||||||
Test item |
316 µg |
12 |
8 |
4.5 |
21 |
19 |
1.5 |
1.0 |
0.9 |
8 |
18 |
||||||||
3 |
19 |
||||||||
Test item |
1000 µg |
7 |
7 |
3.5 |
17 |
17 |
3.5 |
0.9 |
0.8 |
10 |
20 |
||||||||
3 |
13 |
||||||||
Test item |
2500 µg |
5 |
9 |
5.3 |
13 |
8 |
5.0 |
1.2 |
0.4 |
7 |
7 |
||||||||
15 |
3 |
||||||||
Test item |
5000 µg |
4 |
7 |
3.1 |
10 |
11 |
1.2 |
0.9 |
0.5 |
6 |
12 |
||||||||
10 |
10 |
||||||||
4-NODP |
10 µg |
112 |
125 |
11.6 |
/ |
/ |
/ |
17.1 |
/ |
131 |
/ |
||||||||
133 |
/ |
||||||||
2-AA |
2.5 µg |
/ |
/ |
/ |
379 |
423 |
37.8 |
/ |
19.5 |
/ |
443 |
||||||||
/ |
446 |
||||||||
SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn
Mutation factor = mean revertants (test item)
mean revertants (vehicle control)
Tester Strain: TA 102 Experiment 2
Treatment |
Dose/plate |
REVERTANT COLONIES PER PLATE |
MUTATION FACTOR |
||||||
Without activation (-S9) |
With activation (+S9) |
||||||||
Counts |
Mean |
SD |
Counts |
Mean |
SD |
-S9 |
+S9 |
||
A. dest |
|
332 |
364 |
29.3 |
508 |
490 |
21.1 |
1.0 |
1.0 |
389 |
496 |
||||||||
372 |
467 |
||||||||
Test item |
10.0 µg |
293 |
313 |
20.0 |
498 |
518 |
18.8 |
0.9 |
1.1 |
333 |
522 |
||||||||
313 |
535 |
||||||||
Test item |
31.6 µg |
292 |
311 |
37.0 |
565 |
579 |
13.1 |
0.9 |
1.2 |
354 |
581 |
||||||||
288 |
591 |
||||||||
Test item |
100 µg |
347 |
321 |
36.7 |
421 |
466 |
44.4 |
0.9 |
1.0 |
337 |
509 |
||||||||
279 |
469 |
||||||||
Test item |
316 µg |
320 |
324 |
5.1 |
557 |
546 |
12.1 |
0.9 |
1.1 |
323 |
548 |
||||||||
330 |
533 |
||||||||
Test item |
1000 µg |
342 |
328 |
12.1 |
449 |
504 |
55.0 |
0.9 |
1.0 |
324 |
505 |
||||||||
319 |
559 |
||||||||
Test item |
2500 µg |
328 |
332 |
4.6 |
457 |
443 |
14.0 |
0.9 |
0.9 |
337 |
442 |
||||||||
331 |
429 |
||||||||
Test item |
5000 µg |
304 |
337 |
39.1 |
453 |
469 |
31.8 |
0.9 |
1.0 |
380 |
449 |
||||||||
326 |
506 |
||||||||
MMS |
1 µL |
1367 |
1560 |
185.6 |
/ |
/ |
/ |
4.3 |
/ |
1737 |
/ |
||||||||
1577 |
/ |
||||||||
2-AA |
10 µg |
/ |
/ |
/ |
1335 |
1154 |
164.6 |
/ |
2.4 |
/ |
1115 |
||||||||
/ |
1013 |
||||||||
SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn
Mutation factor = mean revertants (test item)
mean revertants (vehicle control)
1. Main Experiment - Toxicity Data, without metabolic activation:
| Test group | Concentration (µg/mL) | Number of Cells 4h after Treatment | Number of Cells 24h after treatment | Number of cells 48h after treatment | SG | RSG (%) | RCE (%) | RTG (%) |
| C1 | 0 | 371000 | 940000 | 1530000 | 14.4 | 100.0 | 100.0 | 100.0 |
| C2 | 0 | 381000 | 1130000 | 1420000 | 16.0 | 100.0 | 100.0 | 100.0 |
| 4 | 100 | 384000 | 1190000 | 1470000 | 17.5 | 115.0 | 121.3 | 139.4 |
| 5 | 250 | 440000 | 890000 | 1660000 | 14.8 | 97.1 | 127.4 | 123.7 |
| 6 | 500 | 374000 | 1010000 | 1500000 | 15.2 | 99.6 | 136.5 | 135.9 |
| 7 | 750 | 332000 | 977000 | 1670000 | 16.3 | 107.2 | 134.1 | 143.8 |
| 8 | 1000 | 356000 | 983000 | 1690000 | 16.6 | 109.2 | 125.3 | 136.8 |
| 9 | 1500 | 310000 | 837000 | 1600000 | 13.4 | 88.0 | 115.6 | 101.7 |
| 10 | 2000 | 345000 | 789000 | 1600000 | 12.6 | 83.0 | 117.4 | 97.4 |
| EMS | 300 | 304000 | 894000 | 1470000 | 13.1 | 86.4 | 90.9 | 78.5 |
| MMS | 10 | 377000 | 967000 | 1660000 | 16.1 | 105.5 | 103.8 | 109.5 |
C: Negative Control; SG: Suspension Growth; RSG: Relative Suspension Growth; RCE: Relative Cloning Efficiency; RTG: Relative Total Growth; EMS: Ethylmethane sulfonate; MMS: Methylmethanesulfonate
2. Main Experiment - Mutagenicity Data, without metabolic activation:
| Cloning Efficiency (CE) | Mutagenicity Data | ||||||||||
| Test Group | Concentration (µg/mL) | Plate 1 | Plate 2 | CE | Number of culture /96 wells | MF (mutants/ 106 cells) | IMF (mutants/ 106 cells) |
||||
| Plate 1 | Plate 2 | Plate 3 | Plate 4 | Mean | |||||||
| C1 | 0 | 73 | 67 | 81.6 | 11 | 19 | 12 | 10 | 13.0 | 89.7 | / |
| C2 | 71 | 72 | 85.4 | 7 | 14 | 10 | 13 | 11.0 | 71.6 | / | |
| 4 | 100 | 70 | 84 | 101.2 | 14 | 17 | 8 | 15 | 13.5 | 75.2 | -5.4 |
| 5 | 250 | 75 | 82 | 106.4 | 11 | 9 | 16 | 13 | 12.3 | 64.4 | -16.3 |
| 6 | 500 | 82 | 79 | 114.0 | 6 | 11 | 11 | 10 | 9.5 | 45.8 | -34.8 |
| 7 | 750 | 77 | 83 | 112.0 | 12 | 9 | 8 | 15 | 11.0 | 54.6 | -26.1 |
| 8 | 1000 | 76 | 80 | 104.6 | 14 | 8 | 16 | 17 | 13.8 | 74.3 | -6.3 |
| 9 | 1500 | 77 | 74 | 96.5 | 13 | 18 | 12 | 18 | 15.3 | 89.9 | 9.3 |
| 10 | 2000 | 76 | 76 | 98.0 | 14 | 10 | 17 | 12 | 13.3 | 76.0 | -4.6 |
| EMS | 300 | 62 | 73 | 75.9 | 71 | 66 | 62 | 66 | 66.3 | 775.6 | 695.0 |
| MMS | 10 | 73 | 71 | 86.6 | 47 | 45 | 45 | 52 | 47.3 | 392.2 | 311.4 |
C: Negative Control; e: Number of culture with cell growth; CE: Cloning efficiency; MF: Mutant Frequency; IMF: Induced Mutant Frequency; EMS: Ethylmethane sulfonate; MMS: Methylmethanesulfonate
3. Main Experiment - Colony Sizing, without metabolic activation:
| Test Group | Concentration (µg/mL) |
Wells with at least 1 colony |
Large colonies | Small colonies | % small colonies |
| C1 | 0 | 52 | 47 | 5 | 9.6 |
| C2 | 44 | 41 | 3 | 6.8 | |
| 8 | 1000 | 55 | 50 | 5 | 9.1 |
| 9 | 1500 | 61 | 51 | 10 | 16.4 |
| 10 | 2000 | 53 | 46 | 7 | 13.2 |
| MMS | 10 | 189 | 112 | 77 | 40.4 |
C: Negative Control; MMS: Methylmethanesulfonate
4. Main Experiment - Toxicity Data, with metabolic activation:
| Test group | Concentration (µg/mL) | Number of Cells 4h after Treatment | Number of Cells 24h after treatment | Number of cells 48h after treatment | SG | RSG (%) | RCE (%) | RTG (%) |
| C1 | 0 | 298000 | 946000 | 1540000 | 14.6 | 100.0 | 100.0 | 100.0 |
| C2 | 341000 | 1060000 | 1430000 | 15.2 | ||||
| 4 | 100 | 326000 | 1040000 | 1530000 | 15.9 | 107.1 | 98.2 | 105.1 |
| 5 | 250 | 286000 | 848000 | 1560000 | 13.2 | 89.0 | 102.9 | 91.6 |
| 6 | 500 | 312000 | 874000 | 1620000 | 14.2 | 95.3 | 104.6 | 99.6 |
| 7 | 750 | 251000 | 759000 | 1600000 | 12.1 | 81.7 | 80.8 | 66.0 |
| 8 | 1000 | 274000 | 708000 | 1730000 | 12.2 | 82.4 | 93.7 | 77.2 |
| 9 | 1500 | 207000 | 432000 | 1660000 | 7.2 | 48.2 | 93.7 | 45.2 |
| 10 | 2000 | 254000 | 596000 | 1630000 | 9.7 | 65.4 | 117.5 | 76.8 |
| B(a)P | 2.5 | 330000 | 755000 | 1580000 | 11.9 | 80.3 | 78.5 | 63.0 |
C: Negative Control; SG: Suspension Growth; RSG: Relative Suspension Growth; RCE: Relative Cloning Efficiency; RTG: Relative Total Growth; B(a)P: Benzo(a)pyrene
5. Main Experiment - Mutagenicity Data, with metabolic activation:
| Cloning Efficiency (CE) | Mutagenicity Data | ||||||||||
| Test Group | Concentration (µg/mL) | Plate 1 | Plate 2 | CE | Number of culture /96 wells | MF (mutants/ 106 cells) | IMF (mutants/106 cells) |
||||
| Plate 1 | Plate 2 | Plate 3 | Plate 4 | Mean | |||||||
| C1 | 0 | 74 | 72 | 89.3 | 6 | 13 | 10 | 12 | 10.3 | 63.5 | / |
| C2 | 75 | 79 | 101.2 | 7 | 7 | 14 | 9 | 9.3 | 50.3 | / | |
| 4 | 100 | 76 | 73 | 93.5 | 9 | 11 | 12 | 18 | 12.5 | 75.0 | 18.1 |
| 5 | 250 | 77 | 75 | 98.0 | 8 | 12 | 13 | 15 | 12.0 | 68.3 | 11.4 |
| 6 | 500 | 79 | 74 | 99.6 | 14 | 19 | 21 | 14 | 17.0 | 98.2 | 41.3 |
| 7 | 750 | 70 | 66 | 77.0 | 11 | 15 | 10 | 9 | 11.3 | 81.2 | 24.3 |
| 8 | 1000 | 69 | 77 | 89.3 | 14 | 18 | 21 | 12 | 16.3 | 104.4 | 47.5 |
| 9 | 1500 | 69 | 77 | 89.3 | 17 | 12 | 17 | 16 | 15.5 | 98.8 | 41.9 |
| 10 | 2000 | 81 | 79 | 112.0 | 13 | 16 | 26 | 22 | 19.3 | 100.9 | 44.0 |
| B(a)P | 2.5 | 64 | 70 | 74.8 | 47 | 46 | 52 | 59 | 51.0 | 511.0 | 454.1 |
C: Negative Control; e: Number of culture with cell growth; CE: Cloning efficiency; MF: Mutant Frequency; IMF: Induced Mutant Frequency; B(a)P: Benzo(a)pyrene
6. Main Experiment - Colony Sizing, with metabolic activation:
| Test Group | Concentration (mM) | Wells with at least 1 colony | Large colonies | Small colonies | % small colonies |
| C1 | 0 | 41 | 40 | 1 | 2.4 |
| C2 | 37 | 37 | 0 | 0.0 | |
| 8 | 1000 | 65 | 48 | 17 | 26.2 |
| 9 | 1500 | 62 | 52 | 10 | 16.1 |
| 10 | 2000 | 77 | 67 | 10 | 13.0 |
| B(a)P | 2.5 | 204 | 115 | 89 | 43.6 |
C: Negative Control; B(a)P: Benzo(a)pyrene
7. Biometry - Main Experiment, without metabolic activation
| Test Group | Concentration (mM) | Mean Mutant Frequency | Mean Induced Mutant Frequency | p-value | Statistical significance |
| C1 | 0 | 89.7 | / | / | / |
| C2 | 71.6 | / | / | / | |
| 4 | 100 | 75.2 | -5.4 | >0.9999 | - |
| 5 | 250 | 64.4 | -16.3 | 0.420 | - |
| 6 | 500 | 45.8 | -34.8 | 0.026 | + |
| 7 | 750 | 54.6 | -26.1 | 0.073 | - |
| 8 | 1000 | 74.3 | -6.3 | 0.925 | - |
| 9 | 1500 | 89.9 | 9.3 | 0.436 | - |
| 10 | 2000 | 76.0 | -4.6 | 0.849 | - |
| EMS | 300 | 775.6 | 695.0 | 0.004 | + |
| MMS | 10 | 392.1 | 311.4 | 0.004 | + |
C: Negative Control; EMS: Ethylmethane sulfonate; MMS: Methylmethanesulfonate; +: significant; -: not significant
8. Biometry - Main Experiment, with metabolic activation:
| Test Group | Concentration (mM) | Mean Mutant Frequency | Mean Induced Mutant Frequency | p-value | Statistical significance |
| C1 | 0 | 63.5 | / | / | / |
| C2 | 50.3 | / | / | / | |
| 4 | 100 | 75.0 | 18.1 | 0.346 | - |
| 5 | 250 | 68.3 | 11.4 | 0.436 | - |
| 6 | 500 | 98.2 | 41.3 | 0.014 | + |
| 7 | 750 | 81.2 | 24.3 | 0.143 | - |
| 8 | 1000 | 104.4 | 47.5 | 0.022 | + |
| 9 | 1500 | 98.8 | 41.9 | 0.022 | + |
| 10 | 2000 | 100.9 | 44.0 | 0.044 | + |
| B(a)P | 2.5 | 511.0 | 454.1 | 0.004 | + |
C: Negative Control; S: Solvent Control; B(a)P: Benzo(a)pyrene; +: significant; -: not significant
Table 1. Summary Experiment I and II, without metabolic activation
|
Dose Group |
Concentration [µg/mL] |
Number of Cells evaluated |
Cytostasis [%] |
Relative Cell Growth [%] |
Micro-nucleated Cells Frequency [%] |
Historical Control Limits Negative Control |
P |
Statistical Significant Increasea |
Exp. I 4 h treatment, 44 h fixation interval |
C |
0 |
2000 |
0 |
100 |
0.80 |
0.35% - 1.33% |
- |
- |
4 |
1000 |
2000 |
0* |
121 |
0.75 |
- |
- |
||
5 |
1500 |
2000 |
0* |
127 |
1.05 |
- |
- |
||
6 |
2000 |
2000 |
0* |
119 |
0.60 |
- |
- |
||
EMS |
1400 |
2000 |
62 |
38 |
3.40 |
- |
+ |
||
Colchicine |
0.8 |
2941 |
63 |
37 |
1.83 |
- |
+ |
||
|
|
|
|
||||||
|
C |
0 |
2000 |
0 |
100 |
0.70 |
0.35% - 1.33% |
- |
- |
5 |
1000 |
2000 |
28 |
72 |
0.50 |
- |
- |
||
6 |
1500 |
2000 |
31 |
69 |
0.55 |
- |
- |
||
7 |
2000 |
2000 |
25 |
75 |
0.55 |
- |
- |
||
EMS |
600 |
2000 |
52 |
48 |
2.15 |
- |
+ |
||
Colchicine |
0.02 |
1444 |
66 |
34 |
1.78 |
- |
+ |
||
C: Negative Control (Culture medium); P: Precipitation (+: precipitation; -: no precipitation); a: statistiscal significant increase compared to negative control (χ2test, p<0.05)
+: significant; - not significant; EMS: Ethylmethanesulfonate, Positive Control (without metabolic activation) [600 µg/mL and 1400 µg/mL]; Colc: ¨psotove Control (without metabolic activation) [0.02µg/mL and0.8 µg/mL]; Relative Cell Growth: 100 x ((CBPITest conc– 1 ) / (CBPIcontrol– 1); Cytostasis [%] = 100- Relative Cell Growth [%]; *: the cytostasis is defined 0, when the relative cell growth exceeds 100%
Table 2. Summary Experiment I, with metabolic activation
|
Dose Group |
Concentration [µg/mL] |
Number of Cells evaluated |
Cytostasis [%] |
Relative Cell Growth [%] |
Micro-nucleated Cells Frequency [%] |
Historical Control Limits Negative Control |
P |
Statistical Significant Increasea |
Exp. I 4 h treatment, 44 h fixation interval |
C |
0 |
2000 |
0 |
100 |
0.45 |
0.32% - 1.53% |
- |
- |
3 |
500 |
2000 |
1 |
99 |
0.75 |
- |
- |
||
5 |
1500 |
2000 |
23 |
77 |
0.35 |
- |
- |
||
6 |
2000 |
2000 |
18 |
82 |
0.25 |
- |
- |
||
CPA |
15 |
2571 |
53 |
47 |
1.55 |
- |
+ |
C: Negative Control (Culture medium); P: Precipitation (+: precipitation; -: no precipitation); a: statistiscal significant increase compared to negative control (χ2test, p<0.05)
+: significant; - not significant; CPA: Cyclophosphamide, Positive Control (with metabolic activation) [15 µg/mL]; Relative Cell Growth: 100 x ((CBPITest conc– 1 ) / (CBPIcontrol– 1); Cytostasis [%] = 100- Relative Cell Growth [%]; *: the cytostasis is defined 0, when the relative cell growth exceeds 100%
Table3. Test for Cytotoxicity, without and with metabolic activation
Dose Group |
Concentration [µg/mL] |
CBPI |
Relative Cell Growth [%] |
Cytostasis [%] |
Precipitate +/- |
without metabolic activation |
|||||
C |
0 |
1.44 |
100 |
0 |
- |
S |
0 |
1.44 |
100 |
0 |
- |
1 |
7.8 |
1.47 |
106 |
0 |
- |
2 |
15.6 |
1.42 |
95 |
5 |
- |
3 |
31.3 |
1.40 |
90 |
10 |
- |
4 |
62.5 |
1.46 |
104 |
0 |
- |
5 |
125 |
1.46 |
105 |
0 |
- |
6 |
250 |
1.44 |
100 |
0 |
- |
7 |
500 |
1.47 |
107 |
0 |
- |
8 |
1000 |
1.44 |
100 |
0 |
- |
9 |
1500 |
1.57 |
129 |
0 |
- |
10 |
2000 |
1.32 |
74 |
26 |
- |
with metabolic activation |
|||||
C |
0 |
1.44 |
100 |
0 |
- |
S |
0 |
1.44 |
100 |
0 |
- |
1 |
7.8 |
1.50 |
114 |
0 |
- |
2 |
15.6 |
1.38 |
88 |
12 |
- |
3 |
31.3 |
1.47 |
107 |
0 |
- |
4 |
62.5 |
1.56 |
128 |
0 |
- |
5 |
125 |
1.46 |
106 |
0 |
- |
6 |
250 |
1.15 |
104 |
0 |
- |
7 |
500 |
1.48 |
110 |
0 |
- |
8 |
1000 |
1.46 |
106 |
0 |
- |
9 |
1500 |
1.60 |
139 |
0 |
- |
10 |
2000 |
1.52 |
120 |
0 |
- |
The CBPI was determined in 500 cells per culture of each test group.
The relative values of the CBPI are related to the negative control.
C: Negative Control (Culture medium); CBPI: Cytokinesis Block Proliferation Index, CBPI = ((c1x 1) + (c2x 2) + (cxx 3))/n; Relative Cell Growth: 100 x ((CBPITest conc– 1) / (CBPIcontrol-1)); c1: mononucleate cells; c2: binucleate cells; cx: multinucleate cells; n: total number of cells; Cytostasis [%] = 100- Relative Cell Growth [%]
the cytostasis is defined 0, when the relative cell growth exceeds 100%
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Ames assay:
In order to investigate the potential of the test item for its ability to induce gene mutations the plate incorporation test with rat liver S9 (experiment I) and the pre-incubation test with hamster liver S9 (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:
Experiment I: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment II: 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
Toxic effects of the test item were noted in some tester strains used in experiment I and II. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Reactive Yellow 42 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.
In vitro micronucleus test:
In order to investigate a possible potential of the test item for its ability to induce micronuclei in human lymphocytes an in vitro micronucleus assay was carried out. The selection of the concentrations was based on data from the pre-experiment. In the main experiment without and with metabolic activation 2000 μg/mL test item was selected as the highest concentration.The following concentrations were evaluated for micronuclei frequencies:
- Experiment I with short-term exposure (4 h):
without metabolic activation: 1000, 1500 and 2000 μg/mL
with metabolic activation: 500, 1500 and 2000 μg/mL
- Experiment II with long-term exposure (44 h):
without metabolic activation: 1000, 1500 and 2000 μg/mL
No precipitate of the test item was noted in all concentration groups evaluated in the main experiment. In experiment I with and without metabolic activation no increase of the cytostasis above 30 % was noted. In experiment II without metabolic activation at a concentration of 1500 μg/mL a cytostasis of 31% was noted but was considered as not biologically relevant due to lack of a dose-response relationship. In experiment I without and with metabolic activation and in experiment II without metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item, nor was an increase in frequency of micronucleated cells observed in any of the experiments.
In conclusion, it can be stated that the test item did not induce structural and/or numerical chromosomal damage in human lymphocytes.
In vitro mouse lymphoma assay:
The test item Reactive Yellow 42 was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The mutant selection cloning was performed by using liquid medium in 96 -microwell plates. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. The test item was investigated at the following concentrations (with and without metabolic activation): 100, 250, 500, 750, 1000, 1500 and 2000 µg/mL.
No precipitation of the test item was observed. In the main experiment with and without metabolic activation the percentage of small colonies in the negative controls and in the highest dose groups treated with the test item was found to be lower than 40%. Thus, all dose groups were considered as not clastogenic.
In conclusion, Reactive Yellow 42 is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Justification for classification or non-classification
Ames test (OECD 471): No biologically relevant increases in revertant colony numbers of any of the five tester strains (TA 98, TA 100, TA 1535, TA 1537 and TA 102) were observed following treatment with the test item at any concentration. Neither inthe presence nor absence of metabolic activation. The test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used and is therefore considered to be non-mutagenic in this bacterial reverse mutation assay.
In vitro micronucleus assay (OECD 487): In two independent experiments (2000 μg/mL,4h exposure, without metabolic activation), no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item. It can therefore be stated the test item did not induce structural and/or numerical chromosomal damage in human lymphocytes.
The test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test.
In vitro mouse lymphoma assay (OECD 490): In the main experiment with and w/o metabolic activation the percentage of small colonies in the negative controls and in the highest dose groups treated with the test item was found to be lower than 40%. Thus, all dose groups were considered as not clastogenic. Based on these test results, Reactive Yellow 42 is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
In conclusion, three in vitro tests, an Ames test, a micronucleus assay and a mammalian cell gene mutation assay were performed to assess the potential genetic toxicity of the test item. The test substance is found to be non-mutagenic in all three performed assays. Hence, based on this information no classification for genotoxicity/mutagenicity is warranted according to the EU Regulation No. 1272/2008, section 3.5.
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