Registration Dossier
Registration Dossier
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EC number: 413-090-5 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Several bacterial reverse mutation tests including the Prival modification with the Substance and structural analogues showed that the Substance does not have any mutagenic properties in bacterial strains. In addition, no genotoxic properties have been seen in the in vivo micronucleus tests in mice and rats. In the in vitro chromosomal aberration assay in CHO cells, a positive effect was seen without S-9 mix at doses which induced precipitations. This is considered a false positive result, as the effects were seen at precipitating concentrations only and the in vivo cytogenetic studies were clearly negative.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2014-04-23 until 2014-07-08
- 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
- 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:
- first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- 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:
- Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
- Test concentrations with justification for top dose:
- Experiment I:
without metabolic activation: 525.0; 1050; 2100; 3150; 4200 µg/mL
with metabolic activation: 1050; 2100; 4200; 6300; 8400 µg/mL
Experiment II:
without metabolic activation: 134.2; 268.4; 536.9; 805.3; 1073.8 µg/mL
with metabolic activation: 268.4; 536.9; 1073.6; 2147.5; 4295.0 µg/mL - Vehicle / solvent:
- deionised water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days
SELECTION AGENT (mutation assays): 6-Thioguanine
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1,5x10exp. 6
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.32 measured in the solvent control versus pH 7.36 measured at 8590 µg/mL)
- Effects of osmolality: no relevant increase (287 measured in the solvent control versus 329 measured at 8590 µg/mL)
- Evaporation from medium: Not examined
- Water solubility:123g/L (without correction for purity)
- Precipitation: No precipitation of the test item was observed up to the maximum concentration in all experiments.
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up or beyond their limit of solubility. Precipitation should be evaluated at the beginning and at the end of treatment by the unaided eye.
The range finding pre-experiment was performed using a concentration range of 67.1 to 8590 µg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24-hours treatment) of metabolic activation. The highest applied concentration in the pre-test on toxicity (8590 µg/ml) was equal to 5 mg/mL of the pure substance.
Relevant toxic effects occurred after 4 hours treatment at 4295 µg/mL and above with metabolic activation. Following 4 hours treatment without metabolic activation toxic effects were noted at 2147.5 µg/mL and above. Another low value of the cloning efficiency was noted at 536.9 µg/mL following 4-hour treatment without metabolic activation. This effect was judged as irrelevant fluctuation rather than a true cytotoxic effect however, as the relative cloning efficiency remained above 50% at the next higher concentration. Following 24 hours treatment without metabolic activation a strong toxic effect occurred at 536.9 µg/mL. At all higher concentrations the cell growth was completely inhibited.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation or phase separation was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treamtment.
There was no relevant shift of the osmolarity and pH value of the medium even at the maximum concentration of the test item.
The dose range of the first experiment was set according to the data generated in the pre-experiment. The dose range of the second experiment was adjusted to data produced in the pre-experiment (without metabolic activation) and in the first experiment (with metabolic activation). The individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at higher concentrations to cover the cytotoxic range more closely.
To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.
COMPARISON WITH HISTORICAL CONTROL DATA: Complies
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 1050 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 805.3 µg/mL and above without metabolic activation and at 4295 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. The difference in cytotoxicity noted in the first and the second experiment with metabolic activation is based on the variability of the cell density during treatment. According to the OECD 476 guideline proliferating cells should be treated so, the actual cell density varies from experiment to experiment. - Conclusions:
- The test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, it is considered to be non-mutagenic in this HPRT assay. - Executive summary:
The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.
The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The main experiments were evaluated at the following concentrations:
exposure
periodS9
mixconcentrations
in µg/mLExperiment I
4 hours
-
525.0
1050
2100
3150
4200
4 hours
+
1050
2100
4200
6300
8400
24 hours
-
134.2
268.4
536.9
805.3
1073.8
4 hours
+
268.4
536.9
1073.6
2147.5
4295.0
No precipitation of the test item was observed up to the maximum concentration in any of the experiments.
Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 1050 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 805.3 µg/mL and above without metabolic activation and at 4295 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. The difference in cytotoxicity noted in the first and the second experiment with metabolic activation is based on the variability of the cell density during treatment. According to the OECD 476 guideline proliferating cells should be treated so, the actual cell density varies from experiment to experiment.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls, the induction factor did not reach or exceed the threshold of 3.0.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.5 up to 23.4 mutants per 106cells; the range of the groups treated with the test item was from 4.5 up to 35.4 mutants per 106 cells.
EMS (150 µg/mL) and DMBA (1.1 µg/mL in experiment I and 2.2 µg/mL in experiment II) were used as positive controls and showed a distinct increase in induced mutant colonies.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- September 2019 to February 2020
- 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)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT). This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid synthesis resulting in the death of TK-competent cells. TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence.
In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK locus (TK+/−) may undergo a single step forward mutation to the TK−/− genotype in which little or no TK activity remains.
The cells used, L5178Y TK+/−, are derived from one of the two clones originated from a thymic tumour induced in a DBA/2 mouse by methylcholanthrene. The use of the TK mutation system in L5178Y mouse lymphoma cells has been well characterised and validated (D. Clive et al., 1979) and is accepted by most of the regulatory authorities.
The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been evaluated that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells. - Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI medium supplemented with Horse serum.
- Properly maintained: yes; Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Prior to use, cells were cleansed of pre-existing mutants. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 tissue fraction: Species: Rat; Strain: Sprague Dawley; Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Numbers: 3512 and 3488
- Test concentrations with justification for top dose:
- Since the test item does not consist of a single active ingredient, the concentration of 5.00 mg/mL was selected as the maximum dose level to be tested as indicated in the Study Protocol. The test item was found to be soluble in complete medium (RPMI 5%).
A cytotoxicity assay was performed, both in the absence and presence of S9 metabolic activation, where the test item was assayed at a maximum dose level of 5.00mg/mL and at a wide range of lower dose levels: 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195mg/mL.
Based on the results obtained in the preliminary trial, three independent assays for mutation at the TK locus were performed using the following dose levels:
Main Assay I (+S9, 3 hour treatment): 5.00, 2.50, 1.25, 0.625 and 0.313 mg/mL.
Main Assay I (-S9, 3 hour treatment): 5.00, 2.50, 1.25, 0.625 and 0.313 mg/mL.
Main Assay III (-S9, 24 hour treatment): 4.00, 2.00, 1.00, 0.500, 0.250 and 0.125 mg/mL. - Vehicle / solvent:
- Test item solutions were prepared using complete medium (RPMI 5%).
- Untreated negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- Cytotoxicity assay
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose level, 0.2mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. Wells containing viable clones were identified by eye using background illumination and then counted.
Mutation assay
The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.
Preparation of test cell cultures was performed as described in section 4.4. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.
In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained, a second experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours).
After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2×105 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.
During the expression period (two days after treatment), the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2×10^5 cells/mL.
Plating for 5-trifluorothymidine resistance: After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/mL) and an estimated 2×103 cells were plated in each well of four 96-well plates. Plateswere incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored. During the scoring of one culture
treated at 0.625mg/mL, one of the four mutation plates was accidentally overturned. This test point was thus excluded from the statistical analysis.
Plating for viability: After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted. - Evaluation criteria:
- For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10^−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance. - Statistics:
- Statistical analysis was performed according to UKEMS guidelines (RobinsonW.D., 1990).
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Survival after treatment:
In the first experiment, in the absence of S9 metabolic activation, moderate toxicity, reducing relative total growth (RTG) to 35% of the concurrent negative control, was noted at 5.00 mg/mL, while no relevant toxicity was noted over the remaining concentrations tested. In the presence of S9 metabolism, test item treatment at 5.00 mg/mL yielded slight toxicity reducing RTG to 69% of the concurrent negative control value, no relevant toxicity was observed over the remaining concentrations tested.
In the second experiment, in the absence of S9 metabolic activation using a long treatment time, dose-related toxicity was observed from 0.500 mg/mL onwards, reducing the RTG values from 61% to 10%. No toxicity was observed at the two remaining concentrations, 0.250 and 0.125mg/mL.
Mutation results:
In Main Assay I, no relevant increases in mutant frequency were observed at any concentration, in the presence of S9 metabolism. A slight increase in mutant frequency was noted in the absence of metabolic activation at the highest concentration tested. However, the observed increase was lower than the Global Evaluation Factor. In addition, no dose-effect relationship was observed, both in the absence and presence of S9 metabolism. In Main Assay II, an increase in mutant frequency was observed at the highest concentration tested and the observed increase was higher than the Global Evaluation Factor. However, the corresponding RTG value was 10% and it is acknowledged that a result cannot be considered positive if the increase in MF occurs only at 10% RTG. In fact, no increases were observed at the other concentrations tested where RTG values were between 31% and 61%.
Furthermore, since the test item is freely soluble at the concentrations tested and is not related to specific chemical classes, positive only by using the long term treatment, the overall results are considered adequate to exclude any mutagenic effect.
Moreover, in the first experiment, it should be noted that unacceptable heterogeneity, due to the absence of one mutation plate (see section 4.6.3), was observed between replicate mutation cultures at 0.625 mg/mL in the presence of S9 metabolism, therefore this concentration was excluded from the statistical analysis.
For the negative and positive controls, the small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. Results are presented in Table 4. An adequate recovery of small colony mutants was observed following treatment with the positive controls. - Conclusions:
- It is concluded that the test substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
- Executive summary:
The test item was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.
Since the test item does not consist of a single active ingredient, the concentration of 5.00 mg/mL was selected as the maximum dose level to be tested as indicated in the Study Protocol. The test item was found to be soluble in complete medium (RPMI 5%).
A cytotoxicity assay was performed, both in the absence and presence of S9 metabolic activation, where the test item was assayed at a maximum dose level of 5.00 mg/mL and at a wide range of lower dose levels: 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 mg/mL.
No precipitation of the test item was noted upon addition of the test item to the cultures and at the end of the 3 and 24 hour treatment periods.
Using the 3 hour treatment time, both in the absence and presence of S9 metabolic activation, no toxicity was observed.
Using the 24 hour treatment time, dose dependent cytotoxicity was noted starting from the highest concentration tested down to 0.625 mg/mL; the Relative Survival was in the range from 11% to 57%. No relevant toxicity was observed over the remaining dose levels tested.
Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the dose levels described in the following table:
Main Assay I (+/-S9, 3 hour treatment): 5.00, 2.50, 1.25, 0.625 and 0.313 mg/mL.
Main Assay II (-S9, 24 hour treatment): 4.00, 2.00, 1.00, 0.500, 0.250 and 0.125 mg/mL.
In the mutation test with the short treatment time in the absence of S9 metabolism, moderate toxicity was noticed at the highest concentration tested reducing the Relative Total Growth (RTG) to 35% of the concurrent negative control. A less pronounced toxic effect was observed in the presence of S9 metabolism, where the test item yielded 69% RTG at the top concentration. Using the long treatment time in the absence of S9 metabolic activation, dose-related toxicity was observed from 0.500 mg/mL onwards, reducing the RTG values from 61% to 10%. No toxicity was observed at the two remaining concentrations tested.
At adequate levels of cytotoxicity, no relevant increases in mutant frequencieswere observed following treatment with the test item, in the absence or presence of S9 metabolism, in any experiment.
Negative and positive control treatments were included in each mutation experiment inthe absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments both as an absolute increase in total mutation frequency (MF) and as an increase in the small colony MF over the concurrent negative control value, indicating the correct functioning of the assay system.
It is concluded that the test item does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 28 July 2011 to 18 August 2011
- 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: M.J. PRIVAL and V.D. MITCHELL: Analysis of a method for testing azo dyes for mutagenicity in Salmonella typhimurium in the presence of flavine mononucleotide and hamster liver S9. Mutation Research 97: 103-116, 1982
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base-pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA tryptophan (trp) reversion system measures trp- to trp+ reversions. The Escherichia coli WP2 uvrA detects mutagens that cause base-pair substitutions (AT to GC).
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- induced rat liver post-mitochondrial fraction (S9 fraction)
uninduced hamster liver post-mitochondrial fraction (S9 fraction) - Test concentrations with justification for top dose:
- No. of concentration Concentration of the test item Concentration (µg/plate)
1 100 mg/mL 5000
2 31.62 mg/mL 1581
3 10 mg/mL 500
4 3.162 mg/mL 158.1
5 1 mg/mL 50
6 0.3162 mg/mL 15.81 - Vehicle / solvent:
- Vehicle: Distilled water
Justification for choice of vehicle:
The solubility of the test item was examined in Distilled water and Dimethyl sulfoxide (DMSO). The test item was soluble in both solvents at 100 mg/mL concentration. Due to the better biocompatibility to the test system, Distilled water was chosen for solvent of the study. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-1,2-phenylene-diamine (NPD), Sodium azide (SAZ), 9-aminoacridine (9AA), Methyl-methanesulfonate (MMS), 2-aminoanthracene (2AA)
- Details on test system and experimental conditions:
- The bacteria used in this assay did not possess the mammalian enzyme system that is known to convert promutagens into mutagenic metabolites. In order to overcome this major drawback, an exogenous metabolic activation system is added in the form of a mammalian microsomal enzyme activation mixture (liver extract, S9 fraction). The activation system uses nicotinamide-adenine dinucleotide phosphate (NADP+)-cytochrome P450 dependent mixed function oxidase enzymes of the liver. The liver extract was obtained from rats, which were pre-treated with phenobarbital and beta-naphthoflavone, two inducers of several drug-metabolizing enzymes.
In addition to the standard Ames test, a modified protocol using pre-incubation with hamster S9 supplemented with flavine mononucleotide was used as proposed by Prival for assessing the mutagenicity of azo-dyes.
Formulation
The behaviour of the test item solutions with the solution of top agar and phosphate buffer was examined in a preliminary solubility test. Distilled water was used as solvent to prepare the stock solution (100 mg/mL) of the test material. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory.
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.
POSITIVE AND NEGATIVE CONTROLS
Positive and negative (solvent) controls were included in the experiments. Routine safety precautions (lab coat, gloves, safety glasses and face mask) were applied to assure personnel health and safety.
Positive Controls
Strain specific positive controls were included in the assay, which demonstrated the effective performance of the test.
Negative Controls (Solvent Controls)
In the study two vehicle control groups were used depending on the solubility of the test item and the solubility of strain specific positive chemicals. The following chemicals were used for vehicle (solvent) control groups:
Dimethyl sulfoxide (DMSO):
Supplier: Sigma-Aldrich Co.
Batch No.: BCBD8784V
Expiry date: December 2016
Grade: puriss p.a., ACS reagent
Purity: ≥99.9%
Distilled water:
Supplier: TEVA Hungary Co.
Batch No.: 0110111
Expiry date: January 2014
Grade: Aqua destillata pro injectione
BACTERIAL STRAINS
Origin
Date of arrival and origin:
Salmonella typhimurium TA98 16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Salmonella typhimurium TA100 04 December 2008, BioReliance Corporation, Rockville, Maryland, USA
Salmonella typhimurium TA1535 16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Salmonella typhimurium TA1537 16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Escherichia coli WP2 uvrA 16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
The true copies of original certificates and other documents of the strains are collected and stored in the Microbiological Laboratory of the CiToxLAB Hungary Ltd.
Genotypes
In addition to histidine or tryptophan mutation, each strain has additional mutations, which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The presence of rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage.
This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair.
Storage
The strains are stored at -80 +/- 10ºC in the Culture Collection of the Microbiological Laboratory of the CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
Confirmation of Phenotypes of Tester Strains
The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture, raw data and reports of phenotype confirmation are stored in the Microbiological Laboratory of CiToxLAB Hungary Ltd.
Spontaneous Reversion of Tester Strains
Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (Escherichia coli WP2 uvrA strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate. Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 1999 to 2010 as follows: Salmonella typhimurium TA98: 9-54, TA100: 58-211, TA1535: 2-31, TA1537: 1-24, Escherichia coli WP2 uvrA: 9-86.
Procedure for Growing Cultures
The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum were used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 degrees C in a Gyrotory water bath shaker.
Viability of the Testing Cultures
The viability of each testing culture was determined by plating 0.1 mL of the 105, 106, 107 and 108 dilutions of cultures on Nutrient Agar (Section 5.4.3.) plates. The viable cell number of the cultures was determined by manual counting.
MEDIA
The Typical Composition (g/1000 mL) of Minimal Glucose Agar:
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0g
di-Potassium hydrogenphosphate 10.0g
Sodium ammonium hydrogenphosphate 3.5g
Agar agar 15.0 g
Distilled water q.s. ad 1000 mL
Minimal glucose agar plates [MERCKOPLATE] (Batch Number: 14518, Expiry date: 15 September 2011) were provided by Merck. Certificates of Analysis were obtained from the Supplier.
Nutrient Broth No.2
Nutrient Broth No.2. 25.0 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
Nutrient Agar
Nutrient Agar 20.0 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
Top Agar for Salmonella typhimurium Strains
Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
Histidine – Biotin solution (0.5 mM):
D-Biotin (F.W. 244.3) 122.2 mg
L-Histidine x HCl x H2O (F.W. 209.63) 104.8 mg
Distilled water q.s. ad 1000 mL
Sterilization was performed by filtration using a 0.22 um membrane filter.
Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100 mL
Agar solution 900 mL
Top Agar for Escherichia coli Strain
Tryptophan solution (2 mg/mL):
L-Tryptophan (F.W. 204.23) 2000 mg
Distilled water q.s. ad 1000 mL
Sterilization was performed by filtration using a 0.22 um membrane filter.
Complete Top Agar for Escherichia coli strain:
Nutrient Broth 50 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution 947.5 mL
METABOLIC ACTIVATION SYSTEM
Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction.
The rat liver post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory in the CiToxLAB Hungary Ltd according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
For azo-dyes and diazo-compounds the modified protocol proposed by Prival and Mitchell is referred to in the OECD guideline No. 471.
This modified protocol differs from the standard plate incorporation assay in five ways:
1. uninduced hamster liver S9 instead of induced rat liver S9 is used
2. the hamster liver S9 mix contains 30% hamster liver extract
3. flavine mononucleotide is added to the S9 mix
4. exogeneous glucose 6-phosphate dehydrogenase, NADH, and four times the standard amount of glucose 6-phosphate is added to the S9 mix
5. a 30 minutes pre-incubation step is used before addition of top agar.
These modifications are needed in order to test the mutagenic potential under conditions in which reduction of the compound to its constituent aromatic amines occurs. The hamster liver post-mitochondrial fraction (S9 fraction) was obtained from Trinova Biochem GmbH.
Rat Liver S9 fraction
Induction of Rat Liver Enzymes
Male Wistar rats (257-305 g, animals were 7-8 weeks old at the initiation of E11041) were treated with phenobarbital (PB) and -naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed.
Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation date of the induction of liver enzymes for preparation S9 used in this study was 07 March 2011 (E11041).
Preparation of Rat Liver Homogenate S9 Fraction
On Day 4, the rats were euthanized and the livers removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000g and the supernatant was decanted and retained. The freshly prepared S9 fraction was distributed in 1-3 mL portions, frozen quickly and stored at -80 10ºC.
Sterility of the preparation was checked.
The protein concentration was determined by colorimetric test by chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of CiToxLAB Hungary Ltd. The protein concentration of the S9 fraction used was determined to be 35.3 g/L. The date of preparation of S9 fraction for this study was 10 March 2011 (CiToxLAB code: E11041).
The biological activity in the Salmonella assay of the batch of S9 used in the study was characterized beside the 2-Aminoanthracene with another mutagen, Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. Each batch of S9 used in this study was found active under the test conditions.
The Rat Liver S9 Mix
Salt solution for S9 mix:
NADP Na 7.66 g
D-glucose-6 phosphate Na 3.53 g
MgCl2 x 6 H2O 4.07 g
KCl 6.15 g
Distilled water q.s. ad 1000.0 mL
The solution was sterilized by filtration through a 0.22 um membrane filter.
The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500.0 mL
Rat liver homogenate (S9) 100.0 mL
Salt solution for S9 mix 400.0 mL
Prior to addition to the culture medium the S9 mix was kept in an ice bath.
Hamster Liver S9 fraction
Hamster Liver Homogenate S9 Fraction
The S9 fraction of Syrian golden hamster liver was provided by Trinova Biochem GmbH (Kerkrader Straße 10; D-35394 Gießen, Germany); Manufacturer: MOLTOX-Molecular Toxicology Inc. (P.O. Box 1189; Boone, North Carolina, 28607 USA). Certificate of Analysis is obtained from the supplier, stored in the Microbiological Laboratory.
Name: Hamster Liver Post Mitochondrial Supernatant (S9)
Supplier: Trinova Biochem GmbH
Manufacturer: MOLTOX Molecular Toxicology Inc.
Catalogue Number: 15-104
Batch Number: 2696
Manufacturing date: 23 December 2010
Expiry date: 23 December 2012
Protein content: 42.5 mg/mL
Storage conditions: -80 ± 10oC
The Hamster liver S9 Mix
The preparation of the S9 Mix was performed according to Prival and Mitchell.
Salt solution for S9 mix:
β-NADP Na 15.31 g
NADH Na2 7.63 g
FMN (Riboflavine-5’-phosphate-sodium salt) 4.78 g
D-glucose-6 phosphate Na 28.20 g
MgCl2 x 6 H2O 8.13 g
KCl 12.31 g
Distilled water ad 1000.0 mL
The solution was sterilized by filtration through a 0.22 um membrane filter.
The complete S9 Mix will be freshly prepared as follows:
Ice cold 0.2 M Sodium phosphate-buffer, pH 7.4 500.0 mL
Hamster liver homogenate (S9) 300.0 mL
Salt solution for S9 mix 200.0 mL
D-glucose-6 phosphate dehydrogenase 3000 U
Prior to addition to the culture medium the S9 mix was kept in an ice bath.
0.2 M Sodium Phosphate Buffer, pH 7.4
Solution A:
Na2HPO4 x 12H2O 71.63 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
Solution B:
NaH2PO4 24.0 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
Sodium phosphate buffer pH 7.4:
Solution A 880 mL
Solution B 120 mL - Evaluation criteria:
- The colony numbers on the untreated /solvent/ positive control and test item treated plates were determined by manual counting. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft Excel TM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions at least two times higher than the reversion rate of the solvent control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains
- the number of reversions at least three times higher than the reversion rate of the solvent control in Salmonella typhimurium TA1535 and TA1537 bacterial strains
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- None
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. In the test each samples (including the controls) were tested in triplicate.
In the Range Finding Test the concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.
The observed numbers of revertant colonies compared to the solvent control were mostly in the normal range in both tester strains with and without metabolic activation. Higher numbers of revertant colonies compared to the solvent control were observed in some cases, but they were well below the relevant threshold value and within the historical control range. Furthermore, no dose-response was observed, therefore, these values were considered as biological variability of the test.
In Salmonella typhimurium TA100 strain, the numbers of revertant colonies on the untreated and negative (solvent) control plates were out of the historical range. However, as the aim of the Preliminary Range Finding test was to get information about toxicity and collect data for dose selection, and the results fulfilled these criteria, the preliminary experiment was considered to be acceptable. - Conclusions:
- The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay including the Prival modification. In this test, the test item had no mutagenic activity on the growth of the applied bacterium tester strains under the test conditions used in this study.
- Executive summary:
The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvr A) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats (Preliminary Range Finding Test and Initial Mutation Test) or from the livers of uninduced hamsters (Confirmatory Mutation Test).
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method, Prival modification).
Based on the results of the Solubility Test, the test item was dissolved in Distilled water. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the two independently performed main experiments (Initial Mutation Test and Confirmatory Mutation Test) were:5000; 1581; 500; 158.1; 50 and 15.81 μg/plate.
In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect.
In all test item treated groups, the numbers of revertant colonies were below the biological relevance when compared with the solvent controls and were within the historical control range and were within the normal biological variability of the test system.
The mean values of revertant colonies of the solvent control plates were within the historical control data range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1999-02-17-1999-03-26
- 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
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Test organism:
Salmonella typhinium strains:
TA98 hisD3052 rfa uvrB + R
TA 100 hisG46 rfa uvrB +R
TA 1535 hisG46 rfa uvrB,
TA 1537 hisC3076 rfa uvrB
and
E. coli WP2 uvrA pkm101 - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- Bacteria:
The strains of Salmonella typhimurium were obtained from professor B.N. Ames, University of California, U.S.A. The strain of E. coli was obtained from the national Collection of Industrial Bacteria, Aberdeen, Scotland.
Bacteria were grown overnight in nutrient broth (25 g Oxid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the cell suspension was checked by nephelometry. Inoculation was performed with stock cultures which had been stored at approx. -80 °C. The different bacterial strains are checked half-yearly with regard to their respective biotin, histidine requirements, membrane permeability, ampicillin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens. All criteria for a valid assay were fulfilled. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9-Mix from induces rat liver and uninduced hamster liver
- Test concentrations with justification for top dose:
- Concentration range (all tests): 50, 160, 500, 1600, and 5000 µg/plate
- Vehicle / solvent:
- Solvent: distilled water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- congo red
- other: 2-aminoanthracene, 1-methyl-3-nitro-1-nitrosoguanidine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation; in suspension;
Test System:
Test Groups: PLATE INCORPORATION TEST
a) Without metabolic activation: 50; 160, 500, 1600 and 5000 µg/plate
b) With metabolic activation (10 % rat liver): 50, 160, 500, 1600 and 5000 µg/plate
PREINCUBATION TEST
a) Without metabolic activation: 50; 160, 500, 1600 and 5000 µg/plate
b) With metabolic activation (30 % rat liver): 50, 160, 500, 1600 and 5000 µg/plate
CONTROL GROUPS:
Negative controls:
a) Untreated control
b: solvent control
Positive controls:
a) without metabolic activation (Sodium-azide for Strain TA 100 and TA 1535, 9 aminoacridine for strain TA 1537, 2-Nitrofluorene for strain 98, MNNG for strain WP2uvrA and 4-NQO for strain WP2uvrA
b): with metabolic activation (10 % rat liver)
2-aminoanthracene for all tester strains
c)-with metabolic activation (30 % Syrian golden hamster liver and preincubation)
2-aminoanthracene for strain TA 100, TA 1535 and TA 1537
Congo red for Strain TA98
Formulation of test compound: dissolved in deionized water at appropriate concentrations immediately before use.
DURATION
- Preincubation period: 30 min preincubation in the presence of 30 % (v/v) Syrian golden hamster S9-mix.
Three volumes of S9 fraction was mixed with 7 volumes of the S9 cofactor solution. - Evaluation criteria:
- Assay considered valid if:
-Solvent control data are within the laboratory´s normal control range for the spontaneous mutant frequency
-Positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory´s normal range
Test compound is classified as mutagenic if has either following effects:
-it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
-it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn If test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratories control range. Test compound proved to be not toxic to the bacterial strains. In all independent mutation tests, the test substance was tested for mutagenicity with concentrations as described in table above. Number of colonies per plate with each strain as well as mean values of 3 plates is given. - Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A pKM 101
- 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
- Effects of pH: NA
- Water solubility:
The test compound did not precipitate on the plates until the highest investigated dose of 5000 µg/plate
The test substance proved to be not toxic to the bacterial strains.
HISTORICAL CONTROL DATA (see table under "overall remarks”) - Conclusions:
- The test substance is not mutagenic in the standard plate test; Ames Test as well as in the preincubation method according to Prival.
- Executive summary:
The substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.
Two independent mutagenicity studies were conducted one standard plate test ( Ames Test) and a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. Additionally, a repeat of the preincubation test was performed with the strain WP2uvrA in the absence of S9 -mix.
For all studies, the compound was dissolved in deionized water, and each bacterial strain was exposed to 5 dose levels. Doses for all studies ranged from 50 to 5000 µg/plate.
Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range and similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies, except in the first preincubation test with the strain WP2uvrA in the absence of S9-mix, where the positive control showed not the expected increase in the number of revertant colonies. This questionable effect was caused by a decomposition of N-Methyl-N-nitrosoguanidine (MNNG). In a repeat of this strain with an alternative positive compound 4 -Nitroquinoline-N-Oxide (4 -NQO) the sensitivity of the assay could be demonstrated.
Toxicity: In the plate incorporation test and also in the preincubation test toxicity was not observed either with or without metabolic activation.
Ames Test:
Mutagenicity: in the absence of the metabolic activation system the test compound did not result in relevant increases in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10 % (v/v)), treatment of the cells with the substance did not result in relevant increases in the number of revertant colonies.
Prival Test:
In the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival the test compound did not cause a significant increases in the number of revertant colonies with any of the tester strains under experimental conditions described.
Therefore, the test substance is not mutagenic in the standard plate test (Ames Test) as well as in the preincubation method according to Prival at the investigated dose levels.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 31 March 1993 to 08 April 1993
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 79/831/EEC
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Principles of method if other than guideline:
- The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Not applicable.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 Mix (rat or hamster)
- Test concentrations with justification for top dose:
- 0, 4, 20, 100, 500, 2500, 10000 ug/plate (both with and without metabolic activation, both Ames and Privall-Mitchell systems)
- Vehicle / solvent:
- Solvent: DMSO
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: without metabolic activation: Sodium-azide: TA 100, TA 1535 9-Aminoacridine: TA 1537 2-Nitrofluorene: TA 98 with rat liver S-9 Mix (10 %): 2-Aminoanthracene: TA 98. TA 100, TA 1535. TA 1537 with hamster liver S-9 Mix (30 %): 2-Aminoanthracene: TA 100.
- Details on test system and experimental conditions:
- Preparation and storage of a liver homogenate fraction ("S-9")
Liver preparations were performed from liver of Aroclor induced Sprague Dawley rats and from non pretreated Syrian hamsters. Male Sprague Dawley rats (200 -300 g) receive a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation is performed at approximatly 0 to 4 °C using cold sterile solution and glassware. The livers from at least 5 - 6 Sprague Daw-ley rats or from 5 - 6 male Syrian golden hamsters (7 - 8 weeks old) are removed and pooled, washed in 150 mM KC1 (approx. 1 ml/g wet livers). The washed livers are cut into small pieces and homogenized in three volumes of KC1. The homogenate is centrifuged at approx. 9000 g for 10 minutes. The supernatant is the S-9 fraction. It is divided into small portions, rapidly frozen and stored at approx. -80 °C for not longer than six months.
Preparation of S-9 Mix
Sufficient S-9 fraction is thawed immediately before each test at room temperature. One or three volumes of S-9 fraction is mixed with nine or seven volumes of the S-9 cofactor solution and kept on ice until used. This preparation is termed S-9 Mix. The concentrations of the different compounds in the S-9 Mix of the rat liver are:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP+
100 mM phosphate buffer pH 7.4
According to the modification proposed by Prival (5) using approx. 30 minutes preincubation in the presence of 30 % Syrian golden hamster S-9 Mix. The S-9 Mix consists of:
8 mM MgCl2
33 mM KCl
20 mM glucose-6-phosphate
2.8 units/ml glucose-6-phosphate dehydrogenase
4 mM NADP+
2 mM NADH
2 mM FMN (Riboflavin-5*-phosphate-Na~salz)
100 mM phosphate buffer pH 7.4
Bacteria
Bacteria are grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The suitable amount of bacteria in the cell suspension is checked by nephelometry. For inoculation, stock cultures which are stored at approx. -80 °C. are used. The compound is tested with the strains Salmonella typhimurium TA 100. TA 1535, TA 1537 and TA 98. Identification of the different bacterial strains is performed periodically and all criteria for a valid assay were achieved as described.
Toxicity experiments and dose range finding
Preliminary toxicity tests were performed with four tester strains using three plates per dose to get information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was evaluated microscopically. In combination with the main experiments, toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 10-6 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).
Mutagenicity test
Two independent experiments for each of the two protocols (Ames, Prival) were performed.
a) - with 10 % rat liver S-9 Mix or buffer and the strains TA 100. TA 1535. TA 1537 and TA 98
Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0.6 % agar, 0.6 I NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. The following ingredients are added (in order) to 2 ml of molten top agar at approximatly 45 °C:
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain 0.1 ml test compound solution 0.5 ml W % rat liver S-9 Mix or buffer
After mixing, the liquid is poured into a petridish with minimal agar (1.5 % agar. Vogel-Bonner E medium with 2 % glucose). After incubation for approximatly 48 hours at approx. 37 °C in the dark, colonies Chis+ revertants) are counted.
b) with 30 % Syrian golden hamster S-9 Mix and preincubation
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 Mix are incu¬bated at approx. 30 °C for the duration of 30 minutes. Subsequently. 2 ml of soft agar which consists of 100 ml agar (0.6 % agar + 0.6 % NaCl) and 10 ml amino-acid solution (minimal amino-acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured on to the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. After incubation for approx. 48 to 72 hours at approx. 37 °C in the dark, colonies (his+ revertants) are counted. - Evaluation criteria:
- Criteria for a positive response
A test article is classified mutagenic if either of the following conditions under a) and b) is achieved:
a test article produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
a test article induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test article at complete bacterial background lawn.
The test results must be reproducible. - Statistics:
- No data
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS
Reaktiv-Gelb F-68 072 FW was tested for mutagenicity with Salmonella typhimurium strains TA 98. TA 100. TA 1535 and TA 1537 in the absence and presence of a metabolic activation systems. S-9 Mix from Sprague Dawley rats (10 % ) and from Syrian golden hamsters (30 %) were used. The results obtained with the test material and positive control compounds are presented in table 1 to 18b. The number of colonies per plate with each strain as well as mean values of 3 plates, corrected to the next whole number are given.
Sterility checks and control plates
Sterility of S-9 Mix and the test compound were indicated by the absence of contamination on the test material and S-9 Mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies.
Toxicity test
The test compound was tested at doses of 4 to 10000 microgram/plate (table 1 -8) and proved to be not toxic to the bacterial strains at doses of 10000 microgram/plate.
For mutagenicity testing 5000 microgram/plate was chosen as the highest dose in the main experiments.
Mutagenicity test with Reaktiv-Gelb F-68 072 FW
Ames-Test:
The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of rat S-9 Mix. No dose dependent effect was obtained.
Prival-Test:
In the presence of hamster liver S-9 Mix (30 %) using the preincubation method according to Prival the test compound did not show any relevant increases in the number of revertant colonies under the experimental conditions described.
It is concluded that Reaktiv-Gelb F-68 072 FW is not mutagenic in the absence and presence of rat S-9 Mix (10 %) using the standard Ames Test procedure. Also in the presence of hamster liver S-9 Mix (30 %) and preincubation the test compound did not induce a significant increase in the number of revertant colonies.
This test was performed according to the methods described. No unforeseen circumstances were observed which have affected the quality and integrity of this study - Conclusions:
- Reaktiv-Gelb F-68 072 FW is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.
- Executive summary:
Study conducted to EU test guidance 79/831/EEC and OCED test guideline 471, in compliance with GLP.
Reaktiv-Gelb F-68 072 FWwas tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium.
The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolising system derived from rat or hamster liver homogenate. A dose range of 6 different doses from 4 microgram/ plate to 5000 microgram/plate was used.
Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.
Toxicity: The test compound proved to be not toxic to the bacterial strains at 10000 microgram/plate.
5000 microgram/plate was chosen as top dose level for the mutagenicity study.
a) Ames Test:
Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10%).treatment of the cells withReaktiv-Gelb F-68 072 FWdid not result in relevant increases in the number of revertant colonies.
b) Prival Test:
In the presence of hamster liver S-9 Mix (30%)using the preincubation method according to PrivalReaktiv-Gelb F-68 072 FWdid not induce a significant increase in the number of revertant colonies, with any of the tester strains.
Summarising, it can be stated that Reaktiv-Gelb F-68 072 FW is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 April 2006 to 25 April 2006
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Sub-line (KI) of Chinese hamster ovary cell line CHO
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CHO KI: Sub-line (KI) of Chinese hamster ovary cell line CHO
ATCCNo.: CCL-61
Lot.No.: 2310352
Supplier: American Type Culture Collection (USA) - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 mix
- Test concentrations with justification for top dose:
- Experiment A with 3/20 h treatment/sampling time
without S9 mix: 1250 μg/ml, 2500 μg/ml and 5000 μg/ml
with S9 mix: 1250 μg/ml, 2500 μg/ml and 5000 μg/ml
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 625 μg/ml, 1250 μg/ml, and 2500 μg/ml
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 1250 μg/ml, 2500 μg/ml and 5000 μg/ml - Vehicle / solvent:
- Ham's F12 medium
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ham's F12 medium
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: Without metabolic activation Ethylmethane sulphonate With metabolic activation N-Nitrosodimethylamine
- Details on test system and experimental conditions:
- Formulation
The test item was prepared in a concentration of 50 mg/ml with Ham's F12 medium (stock solution) at the first step. The necessary amount of test item was weighed into a calibrated volumetric flask. A partial volume of Ham's F12 medium was added and the solution was stirred until homogeneity is reached. Thereafter, Ham's F12 medium was added to the final volume. The appropriate amount of this stock solution was diluted with medium to obtain the examination concentrations. The solution was prepared just before the treatment of the cells. For examined test item concentrations, no precipitation in the medium was noted
Positive Controls
Ethylmethane sulphonate, a known mutagen and chromosome-breaking (clastogenic) agent, was dissolved in Ham's F12 medium and was used as a positive control for the non-activation study at a final concentration of 0.4 ul/ml (Brusick 1989).
N-Nitrosodimethylamine, a clastogen that requires metabolic transformation by microsomal enzymes, was dissolved in Ham's F12 medium and was used as a positive control item for the activation study at a final concentration of 0.4 ul/ml (Brusick 1989).
INDICATOR CELLS
The CHO cell line was originally derived from the ovary of a female Chinese hamster (Puck and Kao, 1967). The CHO KI is a sub-line ofCHO cell line. This cell line has a short doubling time (12-14 h. Preston at al., 1981) and low chromosome number (20-22). The CHO KI cell line was purchased from American Type Culture Collection (USA). The cell stocks are kept in freezer at -80 °C. The laboratory cultures are maintained in 75 cm2 plastic flasks at 37 °C in a humidified atmosphere containing 5 % CO2. The cell cultures are maintained in exponential growth by sub-culturing twice a week. This CHO cell line is mycoplasma free.
Trypsin-EDTA (0.25 % Trypsin, 1 mM EDTAx4Na in Hanks' B.S.S. without Ca2+ and Mg2+) solution was used for cell detachment to subculture.
The detached cells were split back to 3xl05 per 75cm2 plastic flask and fed for 24 hours prior to treatment with fresh medium. The CHO KI cells for this study are grown in Ham's F12 medium supplemented with L-glutamine (1 mM), penicillin (50-100 units/ml), streptomycin (50 ug/ml) and heat-inactivated foetal calf serum (final concentration 10 %). During the 3 hours treatment with test item and positive controls, the serum content was reduced to 0 %. During 20 hours treatment with test item the serum content was reduced to 5 %.
EXTERNAL METABOLIC ACTIVATION SYSTEM
The post-mitochondrial fraction was prepared by the Microbiological Laboratory of LAB International Research Centre Hungary Ltd. according to SOP 007M 09. It is stored at -80 °C and used when needed for studies. The documentation covering the preparation is also stored in the Microbiological Laboratory of LAB International Research Centre Hungary Ltd
PREPARATION OFS9 MIX
The metabolic activation system (S9 mix) was prepared as described by Natarajan et al., (1976) and was set up on the day of use as follows:
S9 fraction 3ml
HEPES* 20mM 2ml
KCl 330mM 1ml
MgCl2 50mM 1ml
NADP** 40mM 1ml
Glucose-6-phosphate 50mM 1ml
Ham'sF12 1ml
* = N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid ** = P-Nicotinamide adenine dinucleotide phosphate
TOXICITY AND DOSE SELECTION
In order to determine the treatment concentrations of test item in the cytogenetic study a dose selection (cytotoxicity assay) was performed. During the cytotoxicity assay 1-3 day old cultures (more than 50 % confluent) were trypsinised and cell suspensions were prepared in Ham's F12 medium. Cells were seeded into 92 x 17 mm dishes (for tissue cultures quality TC sterile) at 5xl05 cells each and were incubated for 24 hours in 10 ml of Ham's F12 medium containing 10 % foetal bovine serum. After 24 hours the cells were treated using increasing concentrations of test item in the absence or presence of S9 mix (100 ul/ml) and were incubated at 37 °C for 3 hours. After treatment the cultures were washed with Ham's F12 medium and covered with Ham's F12 medium containing 10 % foetal bovine serum. Evaluation of cell number was performed at 20 hours (approximately 1.5 normal cell cycles from the beginning of treatment). Another group of cells were treated for 20 hours, without metabolic activation and cell number evaluation was performed at 28 hours. The negative (solvent) control and all treatment groups were run in duplicate. The results of this test were used to select the concentrations of test item for the Chromosome Aberration Assays
CHROMOSOME ABERRATION ASSAYS
The Chromosome Aberration Assays were conducted in two independent experiments in the presence and in the absence of S9 mix.
Experimental Design Cytogenetic Experiment (Experiment A)
3-hour treatment, harvest 20 hours from the beginning of treatment.
GROUPS Concentrations
Solvent Control (H.F12) 5ml/dish
Test item 1250μg/ml
Test item 2500 μg/ml
Test item 5000 μg/ml
Ethylmethane sulphonate 0.4 μl/ml
Solvent Control (H.F12) 4.5 μg/ml + S9mix 100 μg/ml
Test item 1250μg/ml + S9mix 100 μg/ml
Test item 2500 μg/ml + S9mix 100 μg/ml
Test item 5000 μg/ml + S9mix 100 μg/ml
N-Nitrosodimethylamine 0.4 μl/ml + S9mix 100 μg/ml
H.F12=Ham’s F12 medium
The test item was dissolved in Ham's F12 medium for the treatment (stock solution: 50 mg/ml). The appropriate amount of this stock solution was diluted with medium to obtain the examination concentrations. Duplicate cultures were used at each concentration and the negative (solvent) control culture as well as the positive controls for treatment without and with S9 mix. 5 x 105 seeded cells were treated for each group. The culture medium of exponentially growing cell cultures were replaced with serum-free medium containing the test item. The exposure period was 3 hours at 37 °C. Washing the cells with Ham's F12 medium followed the exposure period and then growth medium was added. Sampling was made at 20 hours (approximately 1.5 normal cell cycles from the beginning of treatment). For concurrent measures of cytotoxicity for all treated and negative control cultures 2 (5 x 105) cells were set up. The cytotoxicity assay was performed as described under paragraph Toxicity and Dose Selection.
Experimental Design Cytogenetic Experiment (Experiment B)
3 and 20-hour treatment, harvest 28 hours from the beginning of treatment.
GROUPS Concentrations
Solvent Control (H.F12) 5ml/dish
Test item 1250μg/ml
Test item 2500 μg/ml
Test item 5000 μg/ml
Ethylmethane sulphonate 0.4 μl/ml
Solvent Control (H.F12) 4.5 μg/ml + S9mix 100 μg/ml
Test item 1250μg/ml + S9mix 100 μg/ml
Test item 2500 μg/ml + S9mix 100 μg/ml
Test item 5000 μg/ml + S9mix 100 μg/ml
N-Nitrosodimethylamine 0.4 μl/ml + S9mix 100 μg/ml
H.F12=Ham’s F12 medium
The test item was dissolved in Ham's F12 medium for the treatment (stock solution: 50 mg/ml). The appropriate amount of this stock solution was diluted with medium to obtain the examination concentrations. In the Cytogenetic Experiment B the exposure period without metabolic activation was 20 hours. The exposure period with metabolic activation was 3 hours. Sampling was made at approximately 2 normal cell cycles (28 hours) from the beginning of treatment to cover a potential mitotic delay. Experiment B was, as experiment A, included a concurrent S9 non-activated and S9 activated positive control. For concurrent measures of cytotoxicity for all treated and negative control cultures 2(5x 105) cells were set up. The cytotoxicity assay was performed as described under paragraph Toxicity and Dose Selection.
PREPARATION OF CHROMOSOME
Cell cultures were treated with Colchicine two hours prior to harvesting. After incubation with Colchicine the cells were swollen using 0.075 M KC1 hypotonic solution, then washed three times in fixative, (3:1 mixture of methanol: acetic-acid) until the preparation became plasma free. Chromosomes were dropped onto slides and air-dried.
The preparations were stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies. For control of bias, all slides were coded and scored blind
EXAMINATION OF SLIDES
200 metaphase cells containing 22±2 centromeres were evaluated for structural aberrations from each experimental group.
Chromatid and chromosome type aberrations (gaps, deletions (breaks) and exchanges) were recorded separately. The number of polyploid cells was determined in 100 cells per culture of each test group.
The nomenclature and classification of chromosome aberrations were given based uponlSCN, 1985, and Savage, 1976, 1983. - Evaluation criteria:
- Criteria for a valid assay
The solvent control data are within the laboratory's normal range for the spontaneous mutant frequency.
The positive controls induce increases in the mutation frequency, which are significant.
The test item is regarded as non-clastogenic if:
the number of metaphases with structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data
and/or no significant increase in the number of metaphases with structural chromosome aberration is observed.
A test item is classified as clastogenic if the met of following criteria:
increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (above the range of our historical control data)
the increases are reproducible between replicate cultures and between test (when the treatment conditions are the same)
the increases are statistically significant
Both biological and statistical significance should be considered together - Statistics:
- For statistical analysis, Fisher exact test was utilised.
The parameters evaluated for statistical analysis was as follow: number of cells with aberration (without gaps). - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- SOLUBILITY AND DOSE SELECTION
Reaktiv Gelb F68072 FW was dissolved for treatment in Ham's F12 medium. The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for Chromosome Aberration Assays, both in the absence and in the presence of S9 mix metabolic activation system. Toxicity was determined by assessment of the relative plating efficiency (% relative survival). Three-hour treatment of Reaktiv Gelb F68072 FW in the absence of S9 mix resulted in 100 % relative survival at 625 ug/ml and 86 % relative survival at 5000 ug/ml concentrations. When the three-hour treatment was performed in the presence of metabolic activation the relative survivals were 72 % at the 625 ug/ml concentration and 44 % at 5000 ug/ml concentration. 20-hour treatment of Reaktiv Gelb F68072 FW in the absence of S9 mix caused 82 % relative survival at the 625 ug/ml concentration and 20 % relative survival at the 5000 ug/ml concentration. These results were used to select the concentrations ofReaktiv Gelb F68072 FW for Chromosome Aberration Assays.
CHROMOSOME ABERRATION ASSAY
The cytotoxicity at the highest concentration was adequate in the studies as indicated by available reduction of % cell survival of at least 50 %, except when the three hours treatment of Reaktiv Gelb F68072 FW in the absence of S9 mix resulted 62 % relative survival at 5000 ug/ml (according to the regulations, the limit concentration is 5mg/ml).
In the (solvent) control group the percentage of cells with structural aberration(s) without gap was equal or less than 5 % proving the suitability of the cell line used. The positive controls Ethylmethane sulphonate (0.4 ul/ml) and N-Nitrosodimethylamine (0.4 ul/ml) caused the expected biologically relevant increase of cells with structural chromosome aberrations. The studies were, therefore, considered as valid.
In Experiment A, a three-hour treatment with Reaktiv Gelb F68072 FW in the absence of S9 mix did not cause a biologically significant increase in the number of cells with structural chromosome aberrations at examined concentrations. In this experiment when the test item was examined in the presence of metabolic activation the number the cells with aberrations was a little bit above the historical control at 5000 ug/ml concentration.
In Experiment B, a three-hour treatment with Reaktiv Gelb F68072 FW in the presence of S9 mix caused a statistically significant increase in the number of cells with structural chromosome aberrations at 5000 ug/ml further indicating that the findings in Experiment A were a above a normal biological variation.
When the Reaktiv Gelb F68072 FW was examined (625, 1250, 2500 ug/ml) without S9 mix, with a long treatment period (20 hours) the frequency of the cells with structural chromosome aberrations with and without gaps showed significant and dose-associated increases compared to the concurrent control.
No biologically relevant increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of Reaktiv Gelb F68072 FW. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
positive without activation
In summary, the Reaktiv Gelb F68072 FW tested with a long treatment (20 hours) without metabolic activation induced structural chromosome aberrations in this test in Chinese hamster ovary cells. Therefore, Reaktiv Gelb F68072 FW is considered positive and clastogenic in this system. - Executive summary:
This study has been performed in accordance with the study plan, the OECD Guidelines for Testing of Chemicals No.: 473 and GLP. The report is submitted with a GLP certificate.
The test item, Reaktiv Gelb F68072 FW was tested in a Chromosome Aberration Assay in CHO-KI cells. The test item was dissolved in Ham's F12 medium and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study.
Two independent assays were performed at concentrations and treatment/sampling times as given below:
Experiment Awith 3/20 h treatment/sampling time
without S9 mix: 1250μg/ml, 2500μg/ml and 5000μg/ml
with S9 mix: 1250μg/ml, 2500μg/ml and 5000μg/ml
Experiment Bwith 20/28 h treatment/sampling time
without S9 mix: 625μg/ml, 1250μg/ml, and 2500μg/ml
Experiment Bwith 3/28 h treatment/sampling time
with S9 mix: 1250μg/ml, 2500μg/ml and 5000μg/ml
Two parallel cultures were set up. For each culture, 100 metaphases were scored for structural chromosome aberrations.
In Experiment A, a three-hour treatment with Reaktiv Gelb F68072 FW in the absence of S9 mix did not cause a biologically significant increase in the number of cells with structural chromosome aberrations at examined concentrations. In this experiment when the test item was examined in the presence of metabolic activation the number the cells with aberrations was a little above the historical control range at the 5000μg/ml concentration.
In Experiment B, a three-hour treatment with Reaktiv Gelb F68072 FW in the presence of S9 mix caused a slight increase in the number of cells with structural chromosome aberrations at 5000μg/ml further indicating that the findings in Experiment A were above the range of normal biological variation. When the Reaktiv Gelb F68072 FW was examined (625, 1250, 2500μg/ml) without S9 mix, with a long treatment period (20 hours) the frequency of the cells with structural chromosome aberrations with and without gaps showed significant and dose-associated increases compared to the solvent control.
There was no biologically relevant increase in the rate of polyploid and endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.
The validity of the test was shown by the use of Ethylmethane sulphonate (0.4μl/ml) and N-Nitrosodimethylamine (0.4μl/ml) as positive controls.
In summary, the Reaktiv Gelb F68072 FW tested with a long treatment (20 hours) without metabolic activation induced structural chromosome aberrations in this test in Chinese hamster ovary cells. Therefore, Reaktiv Gelb F68072 FW is considered positive and clastogenic in this system.
Referenceopen allclose all
Summary Table
| relative | relative | relative | mutant | relative | relative | relative | mutant | |||||
| conc. | S9 | cloning | cell | cloning | colonies/ | induction | cloning | cell | cloning | colonies/ | induction | |
| µg/mL | mix | efficiency I | density | efficiency II | 106cells | factor | efficiency I | density | efficiency II | 106cells | factor | |
| % | % | % | % | % | % | |||||||
| Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| Experiment I / 4 h treatment | culture I | culture II | ||||||||||
| Solvent control with water | - | 100.0 | 100.0 | 100.0 | 10.1 | 1.0 | 100.0 | 100.0 | 100.0 | 12.4 | 1.0 | |
| Positive control (EMS) | 150.0 | - | 90.6 | 77.6 | 99.9 | 188.3 | 18.7 | 97.8 | 55.3 | 87.1 | 221.4 | 17.8 |
| Test item | 131.3 | - | 55.9 | culture was not continued# | 79.5 | culture was not continued# | ||||||
| Test item | 262.5 | - | 63.4 | culture was not continued# | 68.7 | culture was not continued# | ||||||
| Test item | 525.0 | - | 49.9 | 93.0 | 111.9 | 8.3 | 0.8 | 67.6 | 69.6 | 105.6 | 4.9 | 0.4 |
| Test item | 1050.0 | - | 29.6 | 93.0 | 104.6 | 5.9 | 0.6 | 38.2 | 81.5 | 93.4 | 12.2 | 1.0 |
| Test item | 2100.0 | - | 32.2 | 95.4 | 90.8 | 21.6 | 2.1 | 31.2 | 91.8 | 100.1 | 19.9 | 1.6 |
| Test item | 3150.0 | - | 9.1 | 83.8 | 96.4 | 4.6 | 0.5 | 16.8 | 76.9 | 96.5 | 5.6 | 0.4 |
| Test item | 4200.0 | - | 8.1 | 76.5 | 94.8 | 17.6 | 1.7 | 10.0 | 66.3 | 85.7 | 21.6 | 1.7 |
| Solvent control with water | + | 100.0 | 100.0 | 100.0 | 16.3 | 1.0 | 100.0 | 100.0 | 100.0 | 7.3 | 1.0 | |
| Positive control (DMBA) | 1.1 | + | 99.7 | 99.9 | 93.8 | 131.5 | 8.1 | 89.2 | 100.6 | 95.2 | 144.6 | 19.7 |
| Test item | 262.5 | + | 82.5 | culture was not continued# | 100.4 | culture was not continued# | ||||||
| Test item | 525.0 | + | 92.9 | culture was not continued# | 69.5 | culture was not continued# | ||||||
| Test item | 1050.0 | + | 81.4 | 120.2 | 76.2 | 4.5 | 0.3 | 63.6 | 130.1 | 50.6 | 6.9 | 0.9 |
| Test item | 2100.0 | + | 89.2 | 109.1 | 76.6 | 18.1 | 1.1 | 78.3 | 107.1 | 93.6 | 19.2 | 2.6 |
| Test item | 4200.0 | + | 89.0 | 121.4 | 107.7 | 9.9 | 0.6 | 59.1 | 93.5 | 125.9 | 16.6 | 2.3 |
| Test item | 6300.0 | + | 69.2 | 107.4 | 120.2 | 16.1 | 1.0 | 60.1 | 97.4 | 129.3 | 13.3 | 1.8 |
| Test item | 8400.0 | + | 62.0 | 94.6 | 100.0 | 9.4 | 0.6 | 47.5 | 88.4 | 99.1 | 11.2 | 1.5 |
| Experiment II / 24 h treatment | culture I | culture II | ||||||||||
| Solvent control with water | - | 100.0 | 100.0 | 100.0 | 5.5 | 1.0 | 100.0 | 100.0 | 100.0 | 23.4 | 1.0 | |
| Positive control (EMS) | 150.0 | - | 95.1 | 84.4 | 98.3 | 450.7 | 82.4 | 96.1 | 83.9 | 78.3 | 639.7 | 27.3 |
| Test item | 33.5 | - | 90.6 | culture was not continued# | 95.7 | culture was not continued# | ||||||
| Test item | 67.1 | - | 93.1 | culture was not continued# | 94.9 | culture was not continued# | ||||||
| Test item | 134.2 | - | 93.1 | 63.8 | 100.8 | 13.6 | 2.5 | 92.5 | 69.5 | 91.5 | 21.9 | 0.9 |
| Test item | 268.4 | - | 95.8 | 67.7 | 108.6 | 8.4 | 1.5 | 84.2 | 50.9 | 89.5 | 9.9 | 0.4 |
| Test item | 536.9 | - | 88.4 | 56.9 | 96.8 | 7.9 | 1.5 | 83.2 | 59.8 | 87.3 | 35.4 | 1.5 |
| Test item | 805.3 | - | 10.3 | 51.9 | 101.3 | 10.0 | 1.8 | 8.8 | 40.7 | 88.4 | 31.4 | 1.3 |
| Test item | 1073.8 | - | 0.0 | 42.0 | 101.5 | 10.8 | 2.0 | 0.0 | 48.8 | 93.2 | 16.5 | 0.7 |
| Experiment II / 4 h treatment | ||||||||||||
| Solvent control with water | + | 100.0 | 100.0 | 100.0 | 17.8 | 1.0 | 100.0 | 100.0 | 100.0 | 20.1 | 1.0 | |
| Positive control (DMBA) | 2.2 | + | 98.9 | 99.6 | 105.1 | 184.1 | 10.3 | 100.3 | 58.2 | 93.6 | 264.7 | 13.2 |
| Test item | 268.4 | + | 87.9 | 86.9 | 96.3 | 19.4 | 1.1 | 105.6 | 104.8 | 99.0 | 20.5 | 1.0 |
| Test item | 536.9 | + | 91.2 | 124.3 | 93.6 | 15.4 | 0.9 | 99.9 | 107.7 | 100.5 | 15.5 | 0.8 |
| Test item | 1073.8 | + | 99.7 | 116.2 | 93.6 | 14.9 | 0.8 | 95.2 | 100.3 | 100.8 | 8.9 | 0.4 |
| Test item | 2147.5 | + | 96.6 | 93.0 | 95.9 | 11.6 | 0.7 | 84.2 | 97.1 | 97.4 | 23.1 | 1.1 |
| Test item | 4295.0 | + | 16.5 | 36.5 | 95.6 | 35.0 | 2.0 | 15.4 | 42.4 | 96.0 | 8.0 | 0.4 |
| Test item | 6442.5 | + | 0.0 | 3.8 | culture was not continued## | 0.0 | 4.5 | culture was not continued## | ||||
| Test item | 8590.0 | + | 0.0 | culture was not continued## | 0.0 | culture was not continued## | ||||||
# culture was not continued since a minimum of only four analysable concentrations is required
## culture was not continued due to exceedingly severe cytotoxic effects
INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test, the plate incorporation method; in the Confirmatory Mutation Test, the pre-incubation method (Prival modification) was used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvr A strain. Each test was performed in the presence and absence of metabolic activation system (±S9 mix) with appropriate untreated, negative (solvent) and positive controls. In the main test each sample (including the controls) were tested in triplicate.
The examined test item concentrations in the main tests were: 5000; 1581; 500; 158.1; 50; and 15.81 /plate.
In the Initial Mutation Test and Confirmatory Mutation Tests, none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.
Using the plate incorporation method, the highest revertant rate was observed in the Initial Mutation Test in Salmonella typhimurium TA98 tester strain without metabolic activation at the concentration of 50 μg/plate. The mutation factor value was 1.97. Higher revertant counts compared to the solvent control were detected for other tested concentrations using this strain. However, no dose-dependence was observed, the observed mutation factor values did not reach the biologically relevant threshold value and the mean numbers of revertant colonies were within the historical control range in all cases.
Using the pre-incubation method (Prival modification), the highest revertant rate was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1537 tester strain without metabolic activation at the concentration of 1581 μg/plate. The mutation factor value was 1.79. Higher revertant counts compared to the solvent control were detected at other tested concentrations in this experiment. However, no dose-response was observed; the observed mutation factor values were below the biologically relevant threshold value and the mean numbers of revertant colonies were within the historical control range. Furthermore, higher number of revertant colonies compared to the Distilled water control was observed for DMSO control (MF: 1.57) also in this experiment
Sporadically, higher numbers of revertant colonies compared to the solvent control were detected in the Initial Mutation Test and Confirmatory Mutation Test in some cases. However, no dose-dependence was observed and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in all cases, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the solvent control were observed in the Initial Mutation Test and Confirmatory Mutation Test at some concentrations. However, the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.
Summary Table of the Confirmatory Mutation Test (Pre-Incubation Method)
Concentrations(ug/plate) |
Mean |
Salmonella typhimuriumtester strains |
Escherichia coli |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvr A |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
20.3 |
37.7 |
133.0 |
144.3 |
9.7 |
11.0 |
3.7 |
8.3 |
29.0 |
44.0 |
MF |
0.98 |
1.07 |
1.60 |
1.18 |
1.32 |
1.22 |
0.79 |
1.19 |
1.19 |
1.27 |
|
DMSO |
Mean |
18.0 |
33.7 |
-- |
115.3 |
-- |
12.7 |
7.3 |
12.0 |
-- |
33.7 |
MF |
0.87 |
0.95 |
-- |
0.94 |
-- |
1.41 |
1.57 |
1.71 |
-- |
0.97 |
|
Distilled water control |
Mean |
20.7 |
35.3 |
83.3 |
122.3 |
7.3 |
9.0 |
4.7 |
7.0 |
24.3 |
34.7 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
5000 |
Mean |
22.0 |
36.3 |
79.3 |
113.7 |
9.0 |
12.0 |
6.0 |
11.3 |
26.7 |
46.0 |
MF |
1.06 |
1.03 |
0.95 |
0.93 |
1.23 |
1.33 |
1.29 |
1.62 |
1.10 |
1.33 |
|
1581 |
Mean |
19.7 |
45.0 |
87.0 |
126.0 |
9.3 |
9.7 |
8.3 |
10.3 |
19.3 |
45.0 |
MF |
0.95 |
1.27 |
1.04 |
1.03 |
1.27 |
1.07 |
1.79 |
1.48 |
0.79 |
1.30 |
|
500 |
Mean |
19.3 |
38.7 |
82.0 |
125.3 |
10.0 |
7.3 |
5.7 |
9.7 |
31.3 |
43.7 |
MF |
0.94 |
1.09 |
0.98 |
1.02 |
1.36 |
0.81 |
1.21 |
1.38 |
1.29 |
1.26 |
|
158.1 |
Mean |
24.0 |
42.0 |
73.0 |
142.3 |
9.3 |
6.3 |
4.0 |
7.7 |
30.3 |
45.7 |
MF |
1.16 |
1.19 |
0.88 |
1.16 |
1.27 |
0.70 |
0.86 |
1.10 |
1.25 |
1.32 |
|
50 |
Mean |
22.0 |
39.0 |
75.3 |
117.3 |
8.3 |
7.3 |
6.7 |
10.7 |
25.7 |
42.0 |
MF |
1.06 |
1.10 |
0.90 |
0.96 |
1.14 |
0.81 |
1.43 |
1.52 |
1.05 |
1.21 |
|
15.81 |
Mean |
20.3 |
43.3 |
86.0 |
139.3 |
9.7 |
8.3 |
4.0 |
6.0 |
19.3 |
37.0 |
MF |
0.98 |
1.23 |
1.03 |
1.14 |
1.32 |
0.93 |
0.86 |
0.86 |
0.79 |
1.07 |
|
NPD (4ug) |
Mean |
293.7 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
MF |
16.31 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
2AA (2ug) |
Mean |
-- |
2301.3 |
-- |
2388.0 |
-- |
210.7 |
-- |
200.7 |
-- |
-- |
MF |
-- |
68.36 |
-- |
20.71 |
-- |
16.63 |
-- |
16.72 |
-- |
-- |
|
2AA (50ug) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
353.3 |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
10.50 |
|
SAZ (2ug) |
Mean |
-- |
-- |
1880.0 |
-- |
1480.0 |
-- |
-- |
-- |
-- |
-- |
MF |
-- |
-- |
22.56 |
-- |
201.82 |
-- |
-- |
-- |
-- |
-- |
|
9AA (50ug) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
486.0 |
-- |
-- |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
66.27 |
-- |
-- |
-- |
|
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1202.7 |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
49.42 |
-- |
|
VALIDITY OF THE TESTS
Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated and solvent control plates were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.
STERILITY CHECKS AND CONTROL PLATES
Sterility of S9-mix and the test material and S9-mix sterility check plates. Control plates gave the expected number of colonies, i.e. values were within the laboratory's historical range.
SOLUBILITY AND TOXICITY
The test compound was dissolved in deionized water and a stock solution of 50 mg/mL was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were used in all experiments.
The test compound proves to be not toxic to the bacterial strains.
MUTAGENICITY:
In all independent mutation tests, the test substance was tested for mutagenicity with the same concentration.
AMES TEST:
The test substance did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or in the presence of rat liver S9-mix in either mutation test. No dose dependant effect was obtained.
PRIVAL TEST:
In the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to prival the test substance did not cause a significant increase in the number of revertant colonies under the experimental conditions described.
All positive controls produced significant increases in the number of revertant colonies. Thus the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.
Main experiment with buffer
Mutability (positive controls) and sterility test of the experiment with Reaktiv-Gelb F-68 072 FW
Number of revertant colonies per plate and mean values using Salmonella typhimurium strains
Strain |
Compound |
Dose μg/plate |
Metab. Activ. |
Mean value |
Colonies per plate |
||
TA100 |
Sodium-azide |
1 |
- |
739 |
748 |
732 |
737 |
TA1535 |
Sodium-azide |
1 |
- |
338 |
356 |
351 |
308 |
TA1537 |
9-Aminoacridine |
50 |
- |
71 |
76 |
64 |
72 |
TA98 |
2-Nitrofluorene |
2.5 |
- |
553 |
543 |
518 |
597 |
|
Reaktiv-Gelb F-68 072 FW |
5000 |
- |
0 |
0 |
0 |
0 |
: absence
Main experiment with 10% rat liver S-9 Mix
Mutability (positive controls) and sterility test of the experiment with Reaktiv-Gelb F-68 072 FW
Number of revertant colonies per plate and mean values using Salmonella typhimurium strains
Strain |
Compound |
Dose μg/plate |
Metab. Active. |
Mean value |
Colonies per plate |
||
TA100 |
2-Aminoanthracen |
0.5 |
+ |
1138 |
1120 |
1178 |
1117 |
TA1535 |
2-Aminoanthracen |
1 |
+ |
129 |
134 |
119 |
134 |
TA1537 |
2-Aminoanthtacen |
1 |
+ |
129 |
145 |
139 |
103 |
TA98 |
2-Aminoanthracen |
0.5 |
+ |
981 |
1017 |
982 |
944 |
|
S-9 mix |
500 μl |
+ |
0 |
0 |
0 |
0 |
|
Reaktiv-Gelb F-68 072 FW |
5000 μg |
+ |
0 |
0 |
0 |
0 |
+ : presence
Main experiment with 30% Syrian golden hamster liver S-9 mix and preincubation
Mutability (positive controls) and sterility test of the experiment with Reaktiv-Gelb F-68 072 FW
Number of revertant colonies per plate and mean values using Salmonella typhimurium strains
Strain |
Compound |
Dose μg/plate |
Metab. Activ. |
Mean value |
Colonies per plate |
||
TA100 |
2-Aminoanthracen |
0.5 |
+ |
648 |
676 |
647 |
622 |
TA1535 |
2-Aminoanthracen |
0.5 |
+ |
112 |
110 |
108 |
118 |
TA1537 |
2-Aminoanthracen |
2.5 |
+ |
334 |
393 |
338 |
270 |
TA98 |
Congored |
500 |
+ |
181 |
108 |
183 |
253 |
TA98 |
Benzidine |
2.14 |
+ |
318 |
334 |
311 |
309 |
|
S-9 mix |
500 μl |
+ |
0 |
0 |
0 |
0 |
|
Reaktiv-Gelb F-68 072 FW |
5000 μg |
+ |
0 |
0 |
0 |
0 |
+ : presence
SUMMARIZED RESULTS OF THE DOSE SELECTION CYTOTOXICITY ASSAY
Test group |
Dose μg/ml |
S9-mix |
Treatment/sampling time |
Cell count dish 1 |
Cell count dish 2 |
Mean value x 104/ml |
Relative survival in percent |
||||
First count |
Second count |
Average |
First count |
Second count |
Average |
||||||
Solvent Control F12 medium |
0 |
- |
3/20 |
86 |
81 |
83.5 |
80 |
84 |
82.0 |
82.75 |
100 |
Reaktiv Gelb F68072 FW |
625 |
- |
3/20 |
85 |
83 |
84.0 |
82 |
81 |
81.50 |
82.75 |
100 |
|
1250 |
- |
3/20 |
70 |
77 |
73.5 |
80 |
79 |
79.5 |
76.60 |
92 |
|
2500 |
- |
3/20 |
75 |
76 |
75.5 |
74 |
71 |
72.5 |
74.00 |
89 |
|
5000 |
- |
3/20 |
72 |
74 |
73.0 |
70 |
68 |
69.0 |
71.00 |
86 |
Solvent Control F12 medium |
0 |
+ |
3/20 |
93 |
86 |
89.5 |
88 |
84 |
86.0 |
87.75 |
100 |
Reaktiv Gelb F68072 FW |
625 |
+ |
3/20 |
64 |
61 |
62.5 |
60 |
66 |
63.0 |
62.75 |
72 |
|
1250 |
+ |
3/20 |
62 |
59 |
60.5 |
63 |
60 |
61.5 |
61.00 |
70 |
|
2500 |
+ |
3/20 |
42 |
48 |
45.0 |
46 |
49 |
47.5 |
46.25 |
53 |
|
5000 |
+ |
3/20 |
39 |
38 |
38.5 |
40 |
36 |
38.0 |
38.25 |
44 |
MEAN PERCENTAGE OF CELLS WITH STRUCTURAL CHROMOSOME ABERRATION(S)
EXPERIMENT A
Concentration (μg/ml) |
S9 mix |
Treatment time |
Harvesting time |
Mean % aberrant cells |
|
incl. gaps |
excl. gaps |
||||
Solvent control (Ham’s F12) |
- |
- |
20 h |
6 |
2 |
Reaktiv Gelb F68072 FW |
|||||
1250 μg/ml |
- |
3 h |
20 h |
7 |
3 |
2500 μg/ml |
- |
3 h |
20 h |
7 |
3 |
5000 μg/ml |
- |
3 h |
20 h |
10 |
5 |
Pos. Control* |
- |
3 h |
20 h |
37 |
25 |
Solvent Control (Ham’s F12) |
+ |
- |
20 h |
9 |
3 |
Reaktiv Gelb F68072 FW |
|||||
1250 μg/ml |
+ |
3 h |
20 h |
10 |
4 |
2500 μg/ml |
+ |
3 h |
20 h |
15 |
5 |
5000 μg/ml |
+ |
3 h |
20 h |
16 |
7 |
Pos. Control** |
+ |
3 h |
20 h |
34 |
24 |
* : Ethylmethane sulphonate (0.4μl/ml)
**: N-Nitrosodimethylamine (0.4 μl/ml)
MEAN PERCENTAGE OF CELLS WITH STRUCTURAL CHROMOSOME ABERRATIONS (S)
EXPERIMENT B
Concentration (μg/ml) |
S9 mix |
Treatment time |
Harvesting time |
Mean % aberrance cells |
|
incl. gaps |
excl. gaps |
||||
Solvent Control (Ham’s F12) |
- |
- |
28 h |
8 |
4 |
Reaktiv Gelb F68072 FW |
|||||
625 μg/ml |
- |
20 h |
28 h |
19 |
9 |
1250 μg/ml |
- |
20 h |
28 h |
29 |
17 |
2500 μg/ml |
- |
20 h |
28 h |
25 |
16 |
Pos. Control* |
- |
20 h |
28 h |
41 |
36 |
Solvent Control (Ham’s F12) |
+ |
- |
28 h |
9 |
3 |
Reaktiv Gelb F68072 FW |
|||||
1250 μg/ml |
+ |
3 h |
28 h |
12 |
4 |
2500 μg/ml |
+ |
3 h |
28 h |
14 |
4 |
5000 μg/ml |
+ |
3 h |
28 h |
21 |
10 |
Pos. Control** |
+ |
3 h |
28 h |
27 |
19 |
* : Ethylmethane sulphonate (0.4 μl/ml)
**: N-Nitrosodimethylamine (0.4 μl/ml)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
The substance did not cause cytogenic effect in the in vivo micronucleus test in mice or rats.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 19 April 1993 to 19 May 1993
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Species: NMRI mouse
Strain: Hoe: NMRKf (SPF71)
Origin: HOECHST AG, Kastengrund, SPF breeding colony
Initial age at test: 7 weeks
Number of animals: 70 (35 males / 35 females)
Bodyweight at start of study: males: x= 28.5 g (23 - 32 g) females: x= 23.7 g (21 - 27 g)
Acclimatization: at least 5 days
Food / water: rat/mice diet Altromin 1324 (Altromin-GmbH,Lage/Lippe), ad libitum
tap water in plastic bottles, ad libitum
Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
Room temperature: 20 ± 3 ⁰C
Relative humidity: approx. 30%
Lighting time: 12 hours daily
Animal identification: fur-marking with KMnO4 and cage numbering - Route of administration:
- oral: gavage
- Vehicle:
- The test compound dilutions were freshly prepared at the day of administration. 5000 mg Reaktiv-Gelb F-68 072 Pi were weight in a mortar, ground well, suspended with starch mucilage, washed out in a 25 ml flask and topped up to the calibration mark. A suspension was formed.
- Details on exposure:
- The test compound was suspended in starch mucilage and dosed once orally at 2000 mg per kg bodyweight to male and female mice
- Duration of treatment / exposure:
- 24, 48 and 72 hours
- Frequency of treatment:
- Single dose
- Post exposure period:
- None
- Remarks:
- Doses / Concentrations:
2000 mg/kg body weight
Basis:
actual ingested - No. of animals per sex per dose:
- Group Dose (mg/kg bwt.) Conc. (%) (w/v) Volume (ml/kg bwt.) Number of animals and sex Killing-time (hours p.a.)
1 0 0 10 5 males/5 females 24
3 2000 20.0 10 5 males/5 females 24
5* 50 0.5 10 5 males/5 females 24
1 0 0 10 5 males/5 females 48
3 2000 20.0 10 5 males/5 females 48
1 0 0 10 5 males/5 females 72
3 2000 20.0 10 5 males/5 females 72
* : Endoxan® (positive control)
Hours p.a. : hours after administration - Control animals:
- yes
- Positive control(s):
- POSITIVE CONTROL: Cyclophosphamide-Endoxan®
For the Endoxan® stock solution, 5 ml distilled water were added to 100 mg Endaxan®in an injection phial and shaken to form a clear solution. The solutions for administration were prepared from this stock solution. For this purpose, 2 ml of the 2 % stock solution were mixed with 6 ml distilled water. - Tissues and cell types examined:
- polychromatic erythrocytes
- Details of tissue and slide preparation:
- Extraction of the bone marrow
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 24, 48 or 72 hours after application. For each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1000 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.
Staining procedure
5 minutes in methanol
5 minutes in May-Grunwalds solution
brief rinsing twice in distilled water
10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Heise)
rinsing in distilled water
drying
coating with Entellan
Evaluation
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group which they belonged to remains unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micro-nuclei occurring in the 1000 normocytes counted, were evaluated statistically. - Evaluation criteria:
- Criteria for a positive response
Both biological and statistical significances are considered together in evaluation.
A substance is considered as positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes for at least one of the time points. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system. - Statistics:
- A Wilcoxon-Test (one-sided) [7,8] is evaluated to check the validity of the study. This is done by comparing the number of polychromatic erythrocytes with micronuclei in the positive control with the negative control.
A Wilcoxon-Test (one-sided) [7,8] is calculated for each measurement group (24h, 48h, 72h) and for polychromatic and normochromatic erythrocytes. These tests are performed sequentially with a multiple level of significance of 5% [5]. Tests on lower dose groups are only performed if the higher dose group is significantly different from the control [6].
The presupposition to make any of the fol1owing comparisons is a difference between the positive control and the negative control (24h). This is tested with a Wilcoxon-Test (two-sided) [7,8] with 5%-level of significance. Wilcoxon-Tests (two-sided) are performed sequentially for the ratio of polychromatic erythrocytes for each measurement group (24h, 48h, 72h) at a multiple level of significance of 5% [5]. Lower dose groups are only tested 1f the higher dose group is significantly different from the control [6]. Actual data were also compared with historical controls. - Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- Mice were treated with 2000 mg Reaktiv-Gelb F-68 072 FW per kg bodyweight to study the induction of micronuclei in bone marrow cells.
All animals survived after application of Reaktiv-Gelb F-68 072 FW. Only orange coloured faeces were observed within 3 hours after application.
The dissection of the animals revealed no test substance related findings.
The bone marrow smears were examined for the occurance of micronuclei in red blood cells.
The incidence of micronucleated polychromatic and normochromatic erythrocytes in the dose groups of Reaktiv-Gelb F-68 072 FW was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes has been observed. The number of normochromatic erythrocytes with micronuclei did not differ from the values of the simultaneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.
Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females indicating the sensitivity of the test system.
Summarizing it can be stated that administration of Reaktiv-Gelb F-68 072 FW did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test. - Conclusions:
- Interpretation of results (migrated information): negative
The results indicate that, under the conditions of the present study, Reaktiv-Gelb F-68 072 FW is not mutagenic in the micronucleus test. - Executive summary:
This test was performed according to EU test guidance 84/449/EEC and OECD test guideline 474. No unforeseen circumstances were observed, which may have affected the quality and integrity of this study. The study was conducted in compliance with the principles of Good Laboratory Practice (GLP).
Reaktiv-Gelb F-68 072 FWwas tested inthemicronucleus test. The test compound was suspended in starch mucilage and dosed once orally at 2000 mg per kg bodyweight to male and female mice, upon the results of the previously conducted dose range finding assay. According to the test procedure the animals were killed 24, 48 or 72 hours after administration.
Endoxan®was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.
The number of polychromatic and normochromatic erythrocytes containing micro-nuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Reaktiv-GelbF-68 072 FW and was statistically not different from the control values.
Endoxan®induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.
The results indicate that, under the conditions of the present study, Reaktiv-Gelb F-68 072 FW is not mutagenic in the micronucleus test.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 22 November 2010 - 19 December 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- This study was performed due to a request from the BAUA (Reference No. 93 04 0620 00) as the animals in the Mouse MNT did not show signs of toxicity and therefore relevant exposure of the target organ (bone marrow) could not be proven.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- micronucleus assay
- Species:
- rat
- Strain:
- other: Rj Han:WI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- EXPERIMENTAL ANIMALS
Species and strain: Rj Han:WI
Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France
Justification of species/strain: In vivo studies in rats with oral gavage (acute toxicity, repeat-dose toxicity, reproduction toxicity) showed that the rat is the relevant rodent species. Unspecific toxic effects were observed in all cited studies, whereas no effects were observed in studies with rats. The rat is regarded as suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity.
Hygienic level: SPF at arrival; standard laboratory conditions during the study
Number of animals:
Preliminary experiment: 6 males + 6 females
3 groups, 2 animals/sex/group
Main test: 35+2 males*
Positive control group: 5 rats
Negative control group: 10 rats
High-dose group: 12 rats
Low- and mid-dose group: 5 rats/group
Age of animals: 10 weeks (preliminary experiment); 8 weeks at the treatment (main test)
Body weight:
361 – 422 g (males, preliminary experiment)
238 – 257 g (females, preliminary experiment)
331 – 377 g (males, main test)
The weight variation did not exceed +/- 20 % of the mean weight/sex at the start of the treatment
Acclimatisation period: At least 5 days
Husbandry
Animal health: Only animals in acceptable health condition were used for the test. Health status was certified by the veterinarian.
Housing/Enrichment: Group caging (5 animals/cage, except the extra animals where 2 animals/cage) to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.
Cage type: III. type polypropylene/polycarbonate
Bedding: Laboratory bedding (Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co. KG; Holzmühle 1, 73494 Rosenberg, Germany)
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature:
21.4 – 22.8°C (preliminary experiment)
20.7 – 23.0°C (main test)
Relative humidity:
34 – 64 % (preliminary experiment)
36 – 52 % (main test)
Ventilation: 15 – 20 air exchanges/hour
Animal room: 245/1 (preliminary experiment); 523 (main test)
The environmental parameters were recorded twice daily during the acclimatisation period and experimental phases of the study.
Food and Water Supply
Animals received ssniff® SM R/M-Z+H H "Autoclavable complete feed for rats and rats – breeding and maintenance" (Batch number: 847 4257, Expiry date: January 2011; and Batch number: 802 4830, Expiry date: May 2011) produced by ssniff Spezialdiäten GmbH (D-59494, Soest, Germany) and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.
The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The contents of the standard diet are detailed in Appendix 8. The supplier provided an analytical certificate for the batch used.
Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). The quality control results are retained in the archives at LAB Research Ltd.
Identification
Animals were identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at LAB Research Ltd. The animal number consisted of 4 digits, the first digit being the group number, the second, 0 for the males (or 1, in case of a replacement), and 5 for the females (or 6, in case of a replacement), and the last 2, the animal number within the group. The cages were marked with identification cards, with information about study code, sex, dose group and individual animal numbers.
Randomisation
The animals were assigned to their respective treatment groups by randomization based on body weights. Animals were randomly allocated to the negative and positive control groups based on the most recent actual body weight; SPSS/PC+ software was used in order to verify homogeneity/variation among/within groups. - Route of administration:
- oral: gavage
- Vehicle:
- Based on the result of a preliminary solubility test, the test item was dissolved in Distilled water for the treatment. The necessary amount of the test item was weighed into a calibrated volumetric flask; the appropriate amount of vehicle was added and stirred to obtain homogenous formulations. The concentration of the test item formulation was chosen to assure the same dosing volumes in rats for all dose levels (10 mL/kg bw). The test item was used for treatment in the main test at concentrations of 200, 100 and 50 mg/mL. The formulations were prepared just before the treatment in the Central Dispensary Unit of LAB Research Ltd.
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study. Stability and homogeneity of test item in distilled water were analytically proven by Analytical Department of LAB International Hungary Research Centre Ltd (05/127-316AN).
Negative (Vehicle) Control Material
Data of the chemical used as vehicle for test item are shown below:
Name:Humaqua (Aqua destillata pro injectione)
Batch No.: 3590210
Source: TEVA Hungary Co.
Expiry date: February 2013
Storage condition: Room temperature - Details on exposure:
- Preliminary toxicity test
A preliminary toxicity test was performed to identify the appropriate maximum dose level for the main test. The preliminary toxicity test also determined whether there are large differences in toxicity between the sexes or not. Groups of two male and female rats were treated on one occasion by oral gavage at the dose levels of 2000, 1000 and 500 mg/kg body weight. The treatment volume was 10 mL/kg body weight. Animals were examined regularly for toxic signs and mortalities. The surviving rats were euthanized 48 hours after treatment. No bone marrow smears were prepared in the preliminary experiment.
Main test
Based on the results of the preliminary toxicity test, doses of 2000, 1000 and 500 mg/kg body weight were examined in the main test. The animals in this Rat Micronucleus Test were treated according to the scheme shown in Table 2, below. - Duration of treatment / exposure:
- 24 / 48 hours; refer to table 2 below
- Frequency of treatment:
- Single dose
- Post exposure period:
- 24 / 48 hours
- Remarks:
- Doses / Concentrations:
500 mg/kg
Basis:
nominal in water - Remarks:
- Doses / Concentrations:
1000 mg/kg
Basis:
nominal in water - Remarks:
- Doses / Concentrations:
2000 mg/kg
Basis:
nominal in water - No. of animals per sex per dose:
- 1. Negative (vehicle)
control -- 10 Males
2. Test item, low dose 500 5 Males
3. Test item, mid dose 1000 5 Males
4. Test item, high dose 2000 10 + 2 Males
5. Positive control
(Cylophosphamide) 25 5 Males
Refer to table 2 below - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Positive Control Material
Cyclophosphamide monohydrate was used as positive control material for the study. It was dissolved in sterile physiological saline solution (0.9% NaCl infusion) for treatment. Routine safety precautions (lab coat, gloves, safety glasses and face mask) for the positive control material were applied to assure personnel health and safety. The positive control formulations were prepared in the Central Dispensary Unit of LAB Research Ltd.
The following chemicals were used for preparing the positive control treatment solution:
Name: Cyclophosphamide monohydrate
Abbreviation: CP
Batch No.: 079K1569
Supplier: Sigma-Aldrich Co.
Expiry date: July 2012
Storage condition: Refrigerated (2-8 °C)
Name: Physiological saline (0.9 % NaCl solution)
Batch No.: 3390210
Source: TEVA Hungary Co.
Expiry date: February 2013
Storage condition: Room temperature - Tissues and cell types examined:
- The mammalian in vivo micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone marrow cells of animals, usually rodents. The purpose of the micronucleus test is to identify substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualisation of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage.
- Details of tissue and slide preparation:
- PREPARATION OF BONE MARROW
The rats from the main test were euthanized by using Euthasol 40 % (pentobarbital sodium for injection) at the scheduled sacrifice.
Deep anaesthesia was confirmed before making incision (death was confirmed by cervical dislocation or by cutting through major cervical blood vessels before discarding carcasses). Bone marrow was obtained from two exposed femurs of rats* immediately after sacrifice.
* Note: No bone marrow smears were prepared from the additional two rats of the high dose group as they were not used as replacement.
The bone marrow was flushed out of each pair of femurs with foetal bovine serum (5 mL) using a syringe and needle into a sterile centrifuge tube. After mixing, the cell suspension was concentrated by a gentle centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides (2 slides / animal). Slides were air-dried at room temperature for approximately 24 hours.
Dried slides were fixed in methanol for a minimum of 5 minutes and allowed to air-dry. Two slides per animal were stained with 10 % Giemsa solution for 20 minutes then thoroughly rinsed with distilled water, and then air-dried at room temperature for at least 12 hours. After staining, coverslips were mounted on them. - Evaluation criteria:
- EXAMINATION OF SLIDES
Prior to microscope analysis the stained slides were given unique code numbers by a person who was not involved in the analysis. The code labels covered all unique identification markings on the slides to ensure that they were scored without bias.
Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field.
The proportion of immature among total (immature + mature) erythrocytes was be determined for each animal by counting a total of at least 1000 cells (immature erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs), in which the number of micronuclei were recorded in both types of erythrocytes.
Criteria for Identification of Micronucleated Erythrocytes:
a) A bluish mauve strongly coloured uniform round or oval particle in the cell.
b) The particle should be large enough for the colour to be recognisable, and it should be located inside the cells. Areas with micronucleus-like particles outside the cells should not be used for analysis.
c) During focusing, the particle should stay uniform in colour /light refraction and shape within a large interval and focus in the same plane as the erythrocyte.
d) The unit of damage is deemed to be the cell, and therefore cells with two or more micronuclei will be counted as single micronucleated cells.
The coded slides were sent for evaluation to Microptic Cytogenetic Services and scored under the control of the Principal Investigator, Natalie Danford, B.Sc., MPH, PhD.
The analysis was conducted in compliance with Good Laboratory Practice as required by the United Kingdom GLP Compliance Regulations 1999 (SI 1999 No. 3106, as amended 2004, SI No. 0994) and which are in compliance with the OECD Principles of Good Laboratory Practice (as revised in 1997) - Statistics:
- According to the Study Plan, statistical analysis will be performed using Kruskal Wallis Non Parametric ANOVA test. However, statistical analysis was not performed for test item treated groups as the frequencies of micronucleated polychromatic erythrocytes in animals in the test groups did not require statistical analysis as the average number of micronuclei/2000 PCE for each dose at both 24h and 48h was less than the vehicle control
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY TOXICITY TEST
In the preliminary solubility test, the test item was soluble in Distilled water at 200 mg/mL concentration. Based on this result and due to technical reason, the following dose groups will be examined in the preliminary experiment: 2000, 1000 and 500 mg/kg body weight (2 animals/sex/group).
In the highest dose group (2000 mg/kg body weight) decreased spontaneous activity, hunched back position, incoordination, splayed gait, discoloured feces were observed for one or more animals on the first day. Splayed gait and/or discoloured feces were observed for these animals 24 hours after the treatment, but the animals were free of clinical signs 48 hours after the treatment.
Based on the results of the preliminary study, dose levels of 2000, 1000 and 500 mg/kg body weight were selected for the micronucleus test. As there were no differences between male and female animals in the preliminary experiment, the main experiment was performed using male rats only.
RAT MICRONUCLEUS TEST
Groups of five male rats were treated with the test item at 2000, 1000 and 500 mg/kg body weight or with the vehicle (Distilled water) in the main experiment. All rats in the vehicle and test item groups were dosed by oral gavage using a dose volume of 10 mL/kg body weight. Animals of the positive control group were treated by intraperitoneal injection with Cyclophosphamide at 25 mg/kg body weight using a dose volume of 2.5 mL/kg body weight.
No marked effect of test item treatment on the body weight of the rats was observed in the main test.
No mortality or systemic clinical signs were observed during the study. In the high dose group (2000 mg/kg body weight) decreased spontaneous activity, splayed gait, incoordination, polierection and discoloured urine were observed for one or more the animals on the first day. Discoloured urine was observed for these animals 24 hours after the treatment, but animals the animals were free of clinical signs 48 hours after the treatment. In the mid dose group (1000 mg/kg body weight) decreased spontaneous activity, splayed gait and piloerection were observed for one or more the animals on the first day, but animals were free of clinical signs 24 hours after the treatment. The animals in the low dose group, furthermore in the negative and positive control group were symptom-free during the whole observation period.
Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. The proportion of immature among total erythrocytes was determined for each animal by also counting mature erythrocytes (NCEs) until a total of 1000 PCEs+NCEs had been observed.
The frequencies of micronucleated polychromatic erythrocytes in animals in the test groups were compared to the values found in the corresponding negative control group. The average numbers of micronuclei in all the treated groups at both 24 and 48h were lower than their corresponding negative controls, so no statistical analysis was appropriate. The positive and negative control results were compared using the Kruskal-Wallis test, and gave a value of H =6.822 (p<0.01). The positive control treatment therefore caused a large, statistically significant increase, demonstrating the sensitivity of the test system.
No indication of an increase in the number of micronucleated normochromatic erythrocytes was observed in the study.
The frequency of micronucleated polychromatic erythrocytes of the negative (solvent) control group was within the range of historical laboratory control data; the positive control item produced a biologically and statistically relevant increase in the number of micronucleated polychromatic erythrocytes; each treated and control group included at least 5 analysable animals; therefore, the test was considered to be valid. - Conclusions:
- No induction of micronuclei in bone marrow erythrocytes was observed following administration of Reactive Yellow F68072 FW to rats at up to and including 2000 mg/kg body weight; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.
- Executive summary:
In the main test, groups of male rats were treated with the vehicle (Distilled water) or the test item at 2000, 1000 and 500mg/kg body weight by oral gavage or the positive control item (Cyclophosphamidedissolved in physiological saline) at 25 mg/kg body weight administered byintraperitoneal injection. Five rats from each group were examined 24 hours after dosing, and a further five rats dosed with the vehicle or test item at 2000mg/kg body weightwere examined 48 hours after dosing. Bone marrow smears were prepared on glass slides for each of the rats, stained, and scored. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells.
No marked effect on body weight was observed in the main test. No mortality or systemic clinical signs were observed during the study. In the high dose group decreased spontaneous activity, splayed gait, incoordination, polierection and discoloured urine were observed for one or more the animals on the first day. Discoloured urine was observed for these animals 24 hours after the treatment, but animals the animals were free of clinical signs 48 hours after the treatment. In the mid dose group decreased spontaneous activity, splayed gait and piloerection were observed for one or more the animals on the first day, but animals were free of clinical signs 24 hours after the treatment. The animals in the low dose group, furthermore in the negative and positive control group were symptom-free during the whole observation period.
The average number of micronuclei/2000 PCE for each dose at both 24h and 48h was less than the vehicle control, and therefore statistical analysis was not appropriate.No biologically or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes were seen in rats treated with the test item, compared to the negative control values. The positive control treatment caused a large, clearly positive response demonstrating the sensitivity of the test system.
It should be noted that the second MNT (oral rat) was performed due to a request from the BAUA (Reference No. 93 04 0620 00) as the animals in the Mouse MNT did not show signs of toxicity and therefore relevant exposure of the target organ (bone marrow) could not be proven.
Referenceopen allclose all
MICRONUCLEUS DATA – Rats sacrificed 24 hours after dosing
Treatment |
Animal Number |
MNPCE/ 2000 PCE |
PCE/1000 PCE+NCE |
Group 1 |
1001 |
3 |
437 |
Negative (vehicle) |
1002 |
2 |
330 |
control |
1003 |
1 |
416 |
(Distilled water) |
1004 |
2 |
280 |
|
1005 |
4 |
384 |
Mean: |
|
2.4 |
369.4 |
SD: |
|
1.14 |
64.22 |
Group 2 |
2001 |
2 |
538 |
Reactive Yellow |
2002 |
2 |
471 |
F68072 FW |
2003 |
0 |
412 |
(500 mg/kg) |
2004 |
2 |
374 |
|
2005 |
2 |
361 |
Mean: |
|
1.6 |
431.2 |
SD: |
|
0.89 |
73.41 |
Group 3 |
3001 |
2 |
359 |
Reactive Yellow |
3002 |
3 |
394 |
F68072 FW |
3003 |
2 |
448 |
(1000 mg/kg) |
3004 |
1 |
378 |
|
3005 |
2 |
385 |
Mean: |
|
2.0 |
392.8 |
SD: |
|
0.7 |
33.43 |
Group 4 |
4001 |
0 |
281 |
Reactive Yellow |
4002 |
2 |
514 |
F68072 FW |
4003 |
2 |
430 |
(2000 mg/kg) |
4004 |
0 |
370 |
|
4005 |
3 |
495 |
Mean: |
|
1.4 |
418.0 |
SD: |
|
1.34 |
95.3 |
Group 5 |
5001 |
21 |
295 |
Positive Control |
5002 |
23 |
294 |
(Cyclophosphamide |
5003 |
21 |
264 |
25 mg/kg) |
5004 |
18 |
262 |
|
5005 |
17 |
249 |
Mean: |
|
20.0 |
272.8 |
SD: |
|
2.5 |
20.63 |
MNPCE: Number of Micronucleated Polychromatic Erythrocytes referring to counts of 2000 PCE.
PCE: Polychromatic Erythrocyte
NCE: Normochromatic Erythrocyte
MICRONUCLEUS DATA – Rats sacrificed 48 hours after dosing
Treatment |
Animal Number |
MNPCE/ 2000 PCE |
PCE/1000 PCE+NCE |
Group 6 |
1006 |
4 |
306 |
Negative (vehicle) |
1007 |
2 |
330 |
control |
1008 |
3 |
396 |
(Distilled water) |
1009 |
1 |
377 |
|
1010 |
5 |
348 |
Mean: |
|
3.0 |
351.4 |
SD: |
|
1.6 |
35.97 |
Group 7 |
4006 |
1 |
361 |
Reactive Yellow |
4007 |
2 |
382 |
F68072 FW |
4008 |
3 |
392 |
(2000 mg/kg) |
4009 |
2 |
298 |
|
4010 |
6 |
369 |
Mean: |
|
2.8 |
360.4 |
SD: |
|
1.92 |
36.86 |
MNPCE: Number of Micronucleated Polychromatic Erythrocytes referring to counts of 2000 PCE.
PCE: Polychromatic Erythrocyte
NCE: Normochromatic Erythrocyte
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Mode of Action Analysis / Human Relevance Framework
The positive effect in the in-vitro chromosome aberration assay in CHO cells is considered to be a false positive result. Kirkland et al (2005) demonstrated an extremely high false-positive rate for in-vitro clastogenicity tests, particularly in mammalian cell tests, when compared to rodent carcinogenicity study results (Poth, A (2008)). “Certain characteristics of the commonly used rodent cell lines (CHO, CHL, V79, L5178Y, etc.), such as their p53 status, their karyotypic instability and their DNA repair deficiencies, are recognized as possibly contributing to the high rate of irrelevant positives. Also the need for exogenous metabolism with the cell systems is expected to contribute to these irrelevant positive findings, as metabolites produced by S9 used as a metabolic source in cell culture may be quite different from those produced by normal human liver metabolism.” Accordingly, “the high false-positive rate of the established in-vitro mammalian cell tests means that an increased number of compounds are subjected to earlier and additional in-vivo genotoxicity testing.”
In addition, it is well known that vinyl-sulphone compounds result in false positive test results in in-vitro tests for clastogenicity (Dearfield KL et al. (1991); Warra TJ et al. (1990)). This is due to the fact that these chemical agents react via the Michael addition reaction. Chemical reactivity via Michael addition is essential for many of the uses for which these compounds are important. Like in the currently assessed dye, vinyl sulphone moieties are used in fiber-reactive dyes (MacGregor et at. (1980)). These compounds are known to deplete glutathione in in‑vitro test systems, in which the concentration of phase II enzymes is very low. Glutathione plays a role in the detoxification of many compounds. Conjugation with glutathione via Michael addition and subsequent excretion is the most common bio-elimination route for these compounds. Since in-vitro systems have low levels of glutathione, the glutathione depletion leads to a positive result in the in-vitro test system, which is not the case in the in-vivo test system, where glutathione is present in adequate amount, as could be shown in the presen in vivo MNT study. Hence, the in-vivo test produces more reliable data for this kind of substance and the substance is not considered to have cytogenic effects.
References
Dearfield KL et al. (1991). Genotoxicity in mouse lymphoma cells .of chemicals capable of Michael addition. Mutagenesis 1991;6(6):519-525
Kirkland et al (2005). Evaluation of the ability of a battery of three in-vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity. Mutat Res. 2005 July 4;584(1–2):1–256
MacGregor et at. (1980). Mutagenicity tests of fabric-finishing agents in Salmonella typhimurium: fiber-reactive wool dyes and cotton flame retardants. Environ. Mutagenesis 1980;2:405-418
Poth, A (2008). Challenges in Testing for Genotoxycity. Genetic Toxicology and Cell Biology, RCC Cytotest Cell Research GmbH
Rothfuss A et al. (2011). Improvement of in-vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing. Mutat Res. 2011 Aug 16;723(2):108-20.
Warra TJ et al. (1990). Methyl vinyl sulphone: A new class of Michael-type genotoxin. Mutat Res. 1990;245:191-199
Additional information
Reactive Yellow F68072 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium according to EU test guidance 79/831/EEC and OCED test guideline 471, in compliance with GLP. Mutagenicity was investigated in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolising system derived from rat or hamster liver homogenate. A dose range of 6 different doses from 4 µg/plate to 5000 µg/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control substances gave the expected increase in the number of revertant colonies.
The test substance proved to be not toxic to the bacterial strains at 10000 µg/plate. Therefore, 5000 µg/plate was chosen as top dose level for the mutagenicity study. In the standard Ames Test, the test substance did not show a dose dependent increase in the number of revertants in any of the bacterial strains either in the absence or in the presence of rat liver activation system (10%). In the presence of hamster liver S-9 Mix (30%) using the preincubation method according to Prival, Reactive Yellow F68072 did not induce a significant increase in the number of revertant colonies, with any of the tester strains.
Summarising, it can be stated that Reactive Yellow F68072 is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.
Structural Analogue 02 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA according to OCED test guideline 471, in compliance with GLP. Two independent mutagenicity studies were conducted one standard plate test (Ames Test) and a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. For all studies, the substance was dissolved in deionized water, and each bacterial strain was exposed to 5 dose levels. Doses for all studies ranged from 50 to 5000 µg/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range and similar to that described in the literature. All the positive control substances gave the expected increase in the number of revertant colonies, except in the first preincubation test with the strain WP2uvrA in the absence of S9-mix, where the positive control showed not the expected increase in the number of revertant colonies. This questionable effect was caused by a decomposition of N-Methyl-N-nitrosoguanidine (MNNG). In a repeat of this strain with an alternative positive substance 4 -Nitroquinoline-N-Oxide (4-NQO) the sensitivity of the assay could be demonstrated.
In the plate incorporation test and also in the preincubation test toxicity was not observed either with or without metabolic activation. In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains either in the absence or in the presence of rat liver activation system (10 % (v/v)). Also in the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival, the test substance did not cause a significant increases in the number of revertant colonies with any of the tester strains.
Therefore, the test substance is not mutagenic in the standard plate test (Ames Test) as well as in the preincubation method according to Prival at the investigated dose levels.
Structural Analogue 03 was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvr A) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats and from the livers of uninduced hamsters. Based on the results of the solubility test, the test substance was dissolved in distilled water. Based on the results of a Range Finding Test, the test substance concentrations in the two independently performed main experiments were 5000; 1581; 500; 158.1; 50 and 15.81 μg/plate. The mean values of revertant colonies of the solvent control plates were within the historical control data range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.
In the plate incorporation (Ames Test) and preincubation method according to Prival, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect. In all test substance treated groups, the numbers of revertant colonies were below the biological relevance when compared with the solvent controls and were within the historical control range and were within the normal biological variability of the test system.
The reported data of this mutagenicity assay show that under the experimental conditions applied the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Hence, the test substance had no mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.
Structural Analogue 03 was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. Since the test substance does not consist of a single active ingredient, the concentration of 5.00 mg/mL was selected as the maximum dose level to be tested. The test substance was found to be soluble in complete medium (RPMI 5%). A cytotoxicity assay was performed, both in the absence and presence of S9 metabolic activation, where the test substance was assayed at a maximum dose level of 5.00 mg/mL and at a wide range of lower dose levels: 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 mg/mL. No precipitation of the test substance was noted upon addition of the test substance to the cultures and at the end of the 3- and 24-hour treatment periods. Using the 3-hour treatment time, both in the absence and presence of S9 metabolic activation, no toxicity was observed. Using the 24-hour treatment time, dose dependent cytotoxicity was noted starting from the highest concentration tested down to 0.625 mg/mL; the Relative Survival was in the range from 11% to 57%. No relevant toxicity was observed over the remaining dose levels tested.
Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the following dose levels:
Main Assay I (+/-S9, 3 hour treatment): 5.00, 2.50, 1.25, 0.625 and 0.313 mg/mL.
Main Assay II (-S9, 24 hour treatment): 4.00, 2.00, 1.00, 0.500, 0.250 and 0.125 mg/mL.
In the mutation test with the short treatment time in the absence of S9 metabolism, moderate toxicity was noticed at the highest concentration tested reducing the Relative Total Growth (RTG) to 35% of the concurrent negative control. A less pronounced toxic effect was observed in the presence of S9 metabolism, where the test substance yielded 69% RTG at the top concentration. Using the long treatment time in the absence of S9 metabolic activation, dose-related toxicity was observed from 0.500 mg/mL onwards, reducing the RTG values from 61% to 10%. No toxicity was observed at the two remaining concentrations tested. At adequate levels of cytotoxicity, no relevant increases in mutant frequencies were observed following treatment with the test substance, in the absence or presence of S9 metabolism, in any experiment.
It is concluded that the test substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
Structural Analogue 01 was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The main experiments were evaluated at the following concentrations:
exposure | S9 | concentrations | ||||
|
| Experiment I | ||||
4 hours | - | 525.0 | 1050 | 2100 | 3150 | 4200 |
4 hours | + | 1050 | 2100 | 4200 | 6300 | 8400 |
|
| |||||
24 hours | - | 134.2 | 268.4 | 536.9 | 805.3 | 1073.8 |
4 hours | + | 268.4 | 536.9 | 1073.6 | 2147.5 | 4295.0 |
No precipitation of the test item was observed up to the maximum concentration in any of the experiments.
Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 1050 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 805.3 µg/mL and above without metabolic activation and at 4295 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. The difference in cytotoxicity noted in the first and the second experiment with metabolic activation is based on the variability of the cell density during treatment. According to the OECD 476 guideline proliferating cells should be treated so, the actual cell density varies from experiment to experiment.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls, the induction factor did not reach or exceed the threshold of 3.0.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.5 up to 23.4 mutants per 106cells; the range of the groups treated with the test item was from 4.5 up to 35.4 mutants per 106 cells. EMS (150 µg/mL) and DMBA (1.1 µg/mL in experiment I and 2.2 µg/mL in experiment II) were used as positive controls and showed a distinct increase in induced mutant colonies.
A micronucleus study in mice was performed according to EU test guidance 84/449/EEC and OECD test guideline 474. Reactive Yellow F68072 was suspended in starch mucilage and dosed once orally at 2000 mg per kg bodyweight to male and female mice, upon the results of the previously conducted dose range finding assay. According to the test procedure the animals were killed 24, 48 or 72 hours after administration. Endoxan® was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.
The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Reactive Yellow F68072 and was statistically not different from the control values.
Endoxan® induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.
The results indicate that, under the conditions of the present study, Reactive Yellow F68072 is not mutagenic in the micronucleus test.
On request of the BAuA, a further micronucleus test with Reactive Yellow F68072 was performed in rats. Groups of male rats were either treated with the vehicle (distilled water) or the test item at 2000, 1000 and 500 mg/kg body weight by oral gavage or the positive control item (cyclophosphamide dissolved in physiological saline) at 25 mg/kg body weight administered by intraperitoneal injection. Five rats from each group were examined 24 hours after dosing, and a further five rats dosed with the vehicle or test item at 2000 mg/kg body weight were examined 48 hours after dosing. Bone marrow smears were prepared on glass slides for each of the rats, stained, and scored. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells.
No marked effect on body weight was observed in the main test. No mortality or systemic clinical signs were observed during the study. In the high dose group decreased spontaneous activity, splayed gait, incoordination, piloerection and discoloured urine were observed for one or more the animals on the first day. Discoloured urine was observed for these animals 24 hours after the treatment, but animals the animals were free of clinical signs 48 hours after the treatment. In the mid dose group decreased spontaneous activity, splayed gait and piloerection were observed for one or more the animals on the first day, but animals were free of clinical signs 24 hours after the treatment. The animals in the low dose group, furthermore in the negative and positive control group were symptom-free during the whole observation period.
The average number of micronuclei/2000 PCE for each dose at both 24h and 48h was less than the vehicle control, and therefore statistical analysis was not appropriate. No biologically or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes were seen in rats treated with the test item, compared to the negative control values. The positive control treatment caused a large, clearly positive response demonstrating the sensitivity of the test system.
Justification for classification or non-classification
The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP an in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds. As the effects are considered adaptive rather than toxicological, no classification is proposed.
The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for prolonged effects is therefore required.
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