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EC number: 241-409-6 | CAS number: 17372-87-1
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
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- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
AMES Assay
Test substance did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
In vitro chromosome aberration study in mammalian cell
The test chemical did not induce chromosomal aberrations in the cultured in the mammalian cell line with and without metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 27-02-2018 - 23-03-2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data is from study report
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Principles of method if other than guideline:
- This study was performed to investigate the potential of Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate (CAS no. 17372-87-1) to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- - Name of the test chemical: Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate
- IUPAC name: disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)benzoate
- Molecular formula: C20H8Br4O5.2Na
- Molecular weight: 691.858 g/mol
- Substance type: Organic - Target gene:
- Histidine
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- other:
- Cytokinesis block (if used):
- No data
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced S9 metabolic activation system
- Test concentrations with justification for top dose:
- 0.0, 0.050, 0.158, 0.501, 1.582 or 5.0 mg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: RO water
- Justification for choice of solvent/vehicle: The test chemical was solulble in RO water - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- RO water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)
DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data
SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data
NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable
NUMBER OF CELLS EVALUATED: No data
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data
OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data
- OTHER: No data - Rationale for test conditions:
- No data
- Evaluation criteria:
- A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant. - Statistics:
- No data
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No precipitation was noted at a dose upto 5 mg/plate in the pre-experiment
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.0, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described for the Trial-I (Plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
In the pre-experiment, the concentration range of the test item was 0.002 – 5 mg/plate based on the solubility and precipitation test. There was no reduction in colony count as well as in background lawn in treated concentrations 5 (T8) mg/plate – 0.002 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.050, 0.158, 0.501, 1.582 and 5 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data - Remarks on result:
- other: No mutagenic potential
- Conclusions:
- Test substance did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
- Executive summary:
Ames assay was performed to investigate the potential of test substance to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0, 0.050, 0.158, 0.501, 1.582, 5.0 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment test substance at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, positive controls were within the range of our historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.
Conclusion
In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Remarks:
- Experimental data from various test chemicals
- Justification for type of information:
- Data for the target chemical is summarized based on the data from various test chemicals
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- other: Refer below principle
- Principles of method if other than guideline:
- WoE derived based on the experimental data from various test chemicals publication.
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- No data
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: McCoy's 5A (modified) medium buffered with 20 mM HEPES and supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: No data - Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- No data
- Metabolic activation:
- with and without
- Metabolic activation system:
- The rat liver microsomal fraction was prepared from Aroclor 1254-induced male Sprague-Dawley rats and was combined with cofactors and culture medium to form the metabolic activation system.
- Test concentrations with justification for top dose:
- 1,Without S9: 0.0, 49.8000, 150.0000, 499.0000 µg/mL
With S9: 0.0, 500.0000, 1500.0000, 5000.0000 µg/mL
2,125,400 and 1250 µg/mL - Vehicle / solvent:
- 1,- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- 1,METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: No data available
- Exposure duration:
Without S9: 8 hrs
With S9: 2 hrs
- Expression time (cells in growth medium):
Without S9: 10-10.5 hrs
With S9: 12 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): Standard harvest time: 10-14 hr after addition of BrdUrd
SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): 5% Giemsa
NUMBER OF REPLICATIONS: Only one trial was performed
NUMBER OF CELLS EVALUATED: 100-200 cells were scored
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available
OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available
OTHER: No data available
2,Details on test system and conditions
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: No data
- Exposure duration: The chemical treatment periods were appoximately 25 hr without S9 and 2 hr with S9.
- Expression time (cells in growth medium): 25-26 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 14 hour
SELECTION AGENT (mutation assays): After staining for 10 min in “concentrated” Hoechst 33258 (5 pg/ml in pH 6.8 buffer) and exposure to “black light” at 55 to 60°C for about 5 min, slides were stained in Giemsa
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data
NUMBER OF REPLICATIONS: No data
NUMBER OF CELLS EVALUATED: 50 cells per dose were scored from the three highest doses
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data
OTHER: Cells were collected by mitotic shake off for evaluation
All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total. ” Gaps and endoreduplications were recorded but were not included in the totals. We did not score aberrations in polyploidy cells but used metaphases with 19-23 chromosomes (the modal number being 21). - Rationale for test conditions:
- No data
- Evaluation criteria:
- Selection of cells for scoring was based on well-spread chromosomes with good morphology and a chromosome number of 21 ± 2. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap,break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomesor cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis.
A positive response at a single dose was designated “ + W”, weak evidence for clastogenicity. If there was a strong trend as the result of a large increase in ABs at a single dose only, we designated the result ‘‘ + W*”. A test was designated “ + ” if at least two doses gave significantly increased responses. - Statistics:
- A binomial sampling assumption as described by Margolin et al. was used to examine absolute increases in ABs over solvent control levels at each dose. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. Only the “total” percent cells with aberrations were analyzed, and a positive response was defined as one for which the adjusted P value was <0.05.
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- (CHO-W-B1)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- Additional information on results
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: Test concentrations for the AB assays were empirically chosen based on toxicity and cell cycle delay as noted in the SCE experiments. At least five
concentrations of the test chemical were selected; the concentrations were spaced using two merged half-log scales (e.g., 1,000, 500, 300, 150, 100, etc.), and the highest concentrations analyzed were those yielding a sufficient number of suitable metaphase cells. The concentrations analyzed generally covered a one-log range.
COMPARISON WITH HISTORICAL CONTROL DATA: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data - Remarks on result:
- other: No mutagenic potential
- Conclusions:
- The test chemical did not induce chromosomal aberrations in the cultured in the mammalian cell line with and without metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
- Executive summary:
Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:
In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. Approximately 24 hr before chemical treatment, cultures were initiated at a density of 1.75 X 106cells/75 cm2flask. In the AB trials without S9, the cultures were treated with the test chemical in medium for 8 hr, washed to remove the test chemical, and treated with colcemid M) for 2-2.5 hr before cell harvest. In the experiments with activation, cultures were exposed to the test chemical in serum free medium with S9 and cofactors for 2 hr, washed to remove the test chemical and S9, and incubated at 37°C with fresh medium for 8 hr. Colcemid was then added, andthe cells were harvested 2 hr later. Thus the total durations of the nonactivated and activated AB experiments were 10hr and 12 hr, respectively, to give 10 hr growth in medium with serum for each experiment. For ABs, slides were stained in 5% Giemsa for 5 min. In early studies, one hundred cells were scored for each ofthree concentrations: the highest test concentration in whichsufficient metaphase cells could be scored and the next two lower concentrations, covering a one-log range. For later studies, 200 cells per dose were scored; however, fewer cells were scored if a test chemical produced a strong positive response or the chemical was toxic. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap,break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomesor cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis. The test chemical did not induce chromosome aberrations in the CHO-LB cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
In another study, in vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was studied at a dose level of 0, 125, 400 and 1250 µg/mL in the absence and presence of S9 using Chinese hamster ovary cells (CHO-W-B1).Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9. 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total”. Gaps and endo-reduplications were recorded but were not included in the totals. Polyploid cells were not scored but used metaphases with 19-23 chromosomes (the modal number being 21). Based on the results noted, the test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.
Based on the data available, the test chemical did not induce chromosomal aberrations in the cultured in the mammalian cell line with and without metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Referenceopen allclose all
TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT
Dose (mg/plate) |
R |
Without metabolic activation (-S9) |
With metabolic activation (+S9) |
||
TA100 |
TA 98 |
TA100 |
TA 98 |
||
NC (0.00) |
R1 |
108 |
22 |
128 |
22 |
R2 |
110 |
24 |
124 |
28 |
|
R3 |
108 |
26 |
126 |
26 |
|
T1 (0.002) |
R1 |
84 |
12 |
92 |
18 |
R2 |
84 |
14 |
84 |
16 |
|
R3 |
92 |
12 |
86 |
16 |
|
T2 (0.005) |
R1 |
86 |
16 |
94 |
20 |
R2 |
90 |
10 |
88 |
22 |
|
R3 |
94 |
14 |
92 |
16 |
|
T3 (0.016) |
R1 |
90 |
16 |
96 |
18 |
R2 |
94 |
14 |
102 |
21 |
|
R3 |
86 |
14 |
102 |
16 |
|
T4 (0.050) |
R1 |
94 |
20 |
104 |
16 |
R2 |
82 |
16 |
106 |
22 |
|
R3 |
96 |
18 |
102 |
18 |
|
T5 (0.158) |
R1 |
100 |
20 |
110 |
20 |
R2 |
92 |
22 |
106 |
20 |
|
R3 |
96 |
20 |
108 |
22 |
|
T6 (0.501) |
R1 |
98 |
22 |
110 |
20 |
R2 |
102 |
20 |
110 |
18 |
|
R3 |
92 |
20 |
112 |
20 |
|
T7 (1.582) |
R1 |
96 |
22 |
114 |
22 |
R2 |
100 |
22 |
110 |
20 |
|
R3 |
98 |
18 |
116 |
18 |
|
T8 (5) |
R1 |
104 |
22 |
114 |
24 |
R2 |
102 |
24 |
112 |
22 |
|
R3 |
102 |
24 |
116 |
24 |
|
PC |
R1 |
1312 |
1072 |
1696 |
968 |
R2 |
1352 |
1024 |
1728 |
912 |
|
R3 |
1376 |
1088 |
1640 |
1016 |
NC = Negative control
PC = Positive control
R = Replicate
T = Test concentration (T8: Highest, T1: Lowest)
4-Nitro-o-phenylenediamine [10μg/plate]: TA 98
Sodium azide [10μg/plate]: TA 100,
2-Aminoanthracene [2.5μg/plate]: TA98, TA100
TABLE 2 - REVERTANT COUNT IN PLATE
INCORPORATION METHOD
(TRIAL I)
Dose (mg/plate) |
R |
In the Presence of Metabolic Activation (+S9) |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
7 |
12 |
22 |
128 |
294 |
R2 |
6 |
12 |
28 |
124 |
284 |
|
R3 |
8 |
14 |
26 |
126 |
276 |
|
T1 (0.050) |
R1 |
5 |
10 |
16 |
104 |
230 |
R2 |
6 |
9 |
22 |
106 |
244 |
|
R3 |
5 |
10 |
18 |
102 |
238 |
|
T2 (0.158) |
R1 |
7 |
10 |
20 |
110 |
232 |
R2 |
5 |
10 |
20 |
106 |
252 |
|
R3 |
5 |
10 |
22 |
108 |
246 |
|
T3 (0.501) |
R1 |
5 |
10 |
20 |
110 |
256 |
R2 |
7 |
12 |
18 |
110 |
232 |
|
R3 |
6 |
10 |
20 |
112 |
244 |
|
T4 (1.582) |
R1 |
6 |
10 |
22 |
114 |
266 |
R2 |
7 |
14 |
20 |
110 |
272 |
|
R3 |
6 |
10 |
18 |
116 |
260 |
|
T5 (5) |
R1 |
7 |
12 |
24 |
114 |
282 |
R2 |
6 |
10 |
22 |
112 |
270 |
|
R3 |
7 |
14 |
24 |
116 |
266 |
|
PC |
R1 |
150 |
436 |
968 |
1696 |
1242 |
R2 |
166 |
452 |
912 |
1728 |
1226 |
|
R3 |
174 |
448 |
1016 |
1640 |
1258 |
Dose (mg/plate) |
R |
In the Absence of Metabolic Activation (-S9) |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
7 |
14 |
22 |
108 |
282 |
R2 |
6 |
12 |
24 |
110 |
296 |
|
R3 |
6 |
14 |
26 |
108 |
288 |
|
T1 (0.050) |
R1 |
5 |
8 |
20 |
94 |
220 |
R2 |
4 |
10 |
16 |
82 |
232 |
|
R3 |
4 |
10 |
18 |
96 |
220 |
|
T2 (0.158) |
R1 |
5 |
12 |
20 |
100 |
228 |
R2 |
4 |
10 |
22 |
92 |
240 |
|
R3 |
6 |
8 |
20 |
96 |
230 |
|
T3 (0.501) |
R1 |
4 |
12 |
22 |
98 |
242 |
R2 |
5 |
10 |
20 |
102 |
254 |
|
R3 |
5 |
10 |
20 |
92 |
236 |
|
T4 (1.582) |
R1 |
6 |
12 |
22 |
96 |
252 |
R2 |
5 |
12 |
22 |
100 |
262 |
|
R3 |
5 |
10 |
18 |
98 |
246 |
|
T5 (5) |
R1 |
6 |
12 |
22 |
104 |
258 |
R2 |
5 |
10 |
24 |
102 |
264 |
|
R3 |
6 |
14 |
24 |
102 |
260 |
|
PC |
R1 |
172 |
1210 |
1072 |
1312 |
1560 |
R2 |
158 |
1230 |
1024 |
1352 |
1588 |
|
R3 |
180 |
1224 |
1088 |
1376 |
1566 |
NC= Negative Control,T=Test concentration (T5: Highest, T1: Lowest),R= Replicate
PC=
Positive
control 2-Aminoanthracene
[2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100
2- Aminoanthracene [10μg/plate]:TA
102 Sodium azide [10μg/plate]:
TA 1535, TA 100
4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate] Methyl methanesulfonate [4μl/plate]: TA 102
TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)
Dose (mg/plate) |
R |
In the Presence of Metabolic Activation (+S9) |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
6 |
12 |
27 |
114 |
256 |
R2 |
5 |
15 |
24 |
116 |
264 |
|
R3 |
7 |
14 |
23 |
110 |
257 |
|
T1 (0.050) |
R1 |
5 |
10 |
21 |
98 |
238 |
R2 |
4 |
12 |
22 |
100 |
243 |
|
R3 |
4 |
12 |
24 |
96 |
232 |
|
T2 (0.158) |
R1 |
5 |
13 |
23 |
102 |
242 |
R2 |
4 |
9 |
21 |
105 |
233 |
|
R3 |
5 |
13 |
21 |
107 |
239 |
|
T3 (0.501) |
R1 |
6 |
10 |
24 |
100 |
246 |
R2 |
5 |
14 |
25 |
106 |
240 |
|
R3 |
4 |
13 |
22 |
110 |
242 |
|
T4 (1.582) |
R1 |
6 |
13 |
23 |
108 |
239 |
R2 |
5 |
11 |
25 |
109 |
245 |
|
R3 |
5 |
14 |
24 |
113 |
254 |
|
T5 (5) |
R1 |
6 |
13 |
25 |
113 |
252 |
R2 |
6 |
12 |
24 |
110 |
249 |
|
R3 |
5 |
14 |
24 |
109 |
247 |
|
PC |
R1 |
176 |
468 |
1406 |
1408 |
1712 |
R2 |
188 |
456 |
1452 |
1386 |
1734 |
|
R3 |
196 |
472 |
1436 |
1362 |
1728 |
Dose (mg/plate) |
R |
In the Absence of Metabolic Activation (-S9) |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
6 |
14 |
25 |
109 |
248 |
R2 |
6 |
12 |
24 |
118 |
254 |
|
R3 |
7 |
14 |
26 |
112 |
262 |
|
T1 (0.050) |
R1 |
5 |
10 |
20 |
98 |
232 |
R2 |
4 |
10 |
22 |
114 |
240 |
|
R3 |
4 |
10 |
24 |
106 |
245 |
|
T2 (0.158) |
R1 |
5 |
10 |
23 |
101 |
238 |
R2 |
4 |
10 |
21 |
110 |
242 |
|
R3 |
5 |
12 |
23 |
113 |
236 |
|
T3 (0.501) |
R1 |
6 |
12 |
24 |
100 |
244 |
R2 |
4 |
12 |
22 |
116 |
239 |
|
R3 |
5 |
10 |
22 |
115 |
236 |
|
T4 (1.582) |
R1 |
5 |
12 |
23 |
108 |
245 |
R2 |
6 |
10 |
21 |
116 |
237 |
|
R3 |
5 |
14 |
25 |
110 |
250 |
|
T5 (5) |
R1 |
6 |
12 |
24 |
112 |
252 |
R2 |
5 |
12 |
23 |
109 |
246 |
|
R3 |
6 |
14 |
25 |
115 |
240 |
|
PC |
R1 |
182 |
1058 |
886 |
1202 |
1528 |
R2 |
194 |
1112 |
904 |
1244 |
1536 |
|
R3 |
206 |
1106 |
938 |
1228 |
1504 |
NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest), R= Replicate
PC=
Positive
control 2-Aminoanthracene
[2.5μg/plate]: TA 1537, TA1535, TA98, TA100
2-Aminoanthracene [10μg/plate]:TA
102 Sodium azide
[10μg/plate]: TA 1535, TA
100,
4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate] Methyl methanesulfonate [4μl/plate]: TA 102
TABLE 4 - MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)
Dose (mg/plate) |
In the presence of Metabolic Activation (+S9) |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
7.00 |
1.00 |
12.67 |
1.15 |
25.33 |
3.06 |
126.00 |
2.00 |
284.67 |
9.02 |
T1 (0.050) |
5.33 |
0.58 |
9.67 |
0.58 |
18.67 |
3.06 |
104.00 |
2.00 |
237.33 |
7.02 |
T2 (0.158) |
5.67 |
1.15 |
10.00 |
0.00 |
20.67 |
1.15 |
108.00 |
2.00 |
243.33 |
10.26 |
T3 (0.501) |
6.00 |
1.00 |
10.67 |
1.15 |
19.33 |
1.15 |
110.67 |
1.15 |
244.00 |
12.00 |
T4 (1.582) |
6.33 |
0.58 |
11.33 |
2.31 |
20.00 |
2.00 |
113.33 |
3.06 |
266.00 |
6.00 |
T5 (5) |
6.67 |
0.58 |
12.00 |
2.00 |
23.33 |
1.15 |
114.00 |
2.00 |
272.67 |
8.33 |
PC |
163.33 |
12.22 |
445.33 |
8.33 |
965.33 |
52.05 |
1688.00 |
44.54 |
1242.00 |
16.00 |
Dose (mg/plate) |
In the Absence of Metabolic Activation (-S9) |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
6.33 |
0.58 |
13.33 |
1.15 |
24.00 |
2.00 |
108.67 |
1.15 |
288.67 |
7.02 |
T1 (0.050) |
4.33 |
0.58 |
9.33 |
1.15 |
18.00 |
2.00 |
90.67 |
7.57 |
224.00 |
6.93 |
T2 (0.158) |
5.00 |
1.00 |
10.00 |
2.00 |
20.67 |
1.15 |
96.00 |
4.00 |
232.67 |
6.43 |
T3 (0.501) |
4.67 |
0.58 |
10.67 |
1.15 |
20.67 |
1.15 |
97.33 |
5.03 |
244.00 |
9.17 |
T4 (1.582) |
5.33 |
0.58 |
11.33 |
1.15 |
20.67 |
2.31 |
98.00 |
2.00 |
253.33 |
8.08 |
T5 (5) |
5.67 |
0.58 |
12.00 |
2.00 |
23.33 |
1.15 |
102.67 |
1.15 |
260.67 |
3.06 |
PC |
170.00 |
11.14 |
1221.33 |
10.26 |
1061.33 |
33.31 |
1346.67 |
32.33 |
1571.33 |
14.74 |
NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation
PC= Positive control
2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100 Methyl methanesulfonate [4μl/plate]: TA 102
2-Aminoanthracene [10μg/plate]:TA 102
Sodium azide [10μg/plate]: TA 1535, TA 100
4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]
TABLE 5 - MEAN REVERTANT COUNT IN
PRE-INCUBATIONMETHOD
(TRIAL II)
Dose (mg/plate) |
In the presence of Metabolic Activation (+S9) |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
6.00 |
1.00 |
13.67 |
1.53 |
24.67 |
2.08 |
113.33 |
3.06 |
259.00 |
4.36 |
T1 (0.050) |
4.33 |
0.58 |
11.33 |
1.15 |
22.33 |
1.53 |
98.00 |
2.00 |
237.67 |
5.51 |
T2 (0.158) |
4.67 |
0.58 |
11.67 |
2.31 |
21.67 |
1.15 |
104.67 |
2.52 |
238.00 |
4.58 |
T3 (0.501) |
5.00 |
1.00 |
12.33 |
2.08 |
23.67 |
1.53 |
105.33 |
5.03 |
242.67 |
3.06 |
T4 (1.582) |
5.33 |
0.58 |
12.67 |
1.53 |
24.00 |
1.00 |
110.00 |
2.65 |
246.00 |
7.55 |
T5 (5) |
5.67 |
0.58 |
13.00 |
1.00 |
24.33 |
0.58 |
110.67 |
2.08 |
249.33 |
2.52 |
PC |
186.67 |
10.07 |
465.33 |
8.33 |
1431.33 |
23.35 |
1385.33 |
23.01 |
1724.67 |
11.37 |
Dose (mg/plate) |
In the Absence of Metabolic Activation (-S9) |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
6.33 |
0.58 |
13.33 |
1.15 |
25.00 |
1.00 |
113.00 |
4.58 |
254.67 |
7.02 |
T1 (0.050) |
4.33 |
0.58 |
10.00 |
0.00 |
22.00 |
2.00 |
106.00 |
8.00 |
239.00 |
6.56 |
T2 (0.158) |
4.67 |
0.58 |
10.67 |
1.15 |
22.33 |
1.15 |
108.00 |
6.24 |
238.67 |
3.06 |
T3 (0.501) |
5.00 |
1.00 |
11.33 |
1.15 |
22.67 |
1.15 |
110.33 |
8.96 |
239.67 |
4.04 |
T4 (1.582) |
5.33 |
0.58 |
12.00 |
2.00 |
23.00 |
2.00 |
111.33 |
4.16 |
244.00 |
6.56 |
T5 (5) |
5.67 |
0.58 |
12.67 |
1.15 |
24.00 |
1.00 |
112.00 |
3.00 |
246.00 |
6.00 |
PC |
194.00 |
12.00 |
1092.00 |
29.60 |
909.33 |
26.41 |
1224.67 |
21.20 |
1522.67 |
16.65 |
NC= Negative Control, T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation
PC= Positive control
2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100
2-Aminoanthracene [10μg/plate]: TA 102
Sodium azide [10μg/plate]: TA 1535, TA 100
4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]
Methyl methanesulfonate: [4μl/plate]: TA 102
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Data for test chemicals was reviewed to determine the mutagenic nature of disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)benzoate (17372-87-1). The studies are as mentioned below:
AMES Assay
Ames assay was performed to investigate the potential of test substance to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0, 0.050, 0.158, 0.501, 1.582, 5.0 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment test substance at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, positive controls were within the range of our historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.
Conclusion
In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.
Test substance was tested for mutagenic potential with the Salmonella/ mammalian-microsome test. The 2 ml of liquid top agar was cooled to 45°C and 0.1 ml of a broth culture of microorganism and test substance at a dose level of 10 -250 mg in volumes of ≤ 0.4 ml of DMSO was added prior to placing on minimal agar plates. After 48 h incubation at 37°C, the colonies which reverted to the prototroph were counted and compared to counts on the control plate (containing no test substance) to demonstrate mutagenicity or toxicity. Materials which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, were denoted as mutagens. Those which reduced the number of revertants were considered inhibitory. The test compound did not induce mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535 and hence is negative for gene mutation in vivo.
Salmonella/ mammalian-microsome test (Spot test) was performed to evaluate the mutagenic nature of the test compound. The spot test was used to screen the test material for potential mutagenicity. The test material was placed in the center of the plate. Eosin was tested with and without the S9 mix. Inhibition of the bacterium was indicated by a clearing of the background lawn in a zone surrounding the test material. Mutagenicity was indicated by a clustering of revertant colonies directly around the test material or at the edge of the inhibitory zone. A known mutagen, Captan, was used as a reference mutagen. The test compound did not induce mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535) in the spot test performed and hence is negative for gene mutation in vitro.
Gene mutation toxicity study was performed to determine the mutagenic nature of test substance. The study was performed using Salmonella typhimurium strains TA98, TA97, TA100, TA1537 and TA1535 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in water as solvent and used at dose levels 0, 100, 333, 666, 1000, 1666, 3333, 6666, 10000, 15000 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. The test compound did not induce mutation in the Salmonella typhimurium tester strains TA100, TA1537 and TA98 in the presence and absence of S9 metabolic activation system. It however induced gene mutation in the tester strains TA1535 and TA97 in the presence of S9 metabolic activation system.
Salmonella/mammalian microsome assay was performed to evaluate the mutagenic nature of the test compound. The dye was dissolved in test substance and up to 0.2 ml was introduced into 2.5 ml of the tempered top agar together with 0.1 ml Salmonella typhimurium broth suspension and 0.25 ml Aroclor 1254 induced rat liver S9. The mixtures was plated on 20 ml of Vogel-Bonner E bottom agar in the usual fashion and incubated for 3 days at 35°. Each agent was tested with all 5 basic tester strains (TA1535, TA100, TA1537, TA1538, TA98) with and without microsomal activation at 3 or more concentrations (0, 50, 100 or 500 µg/plate). The test compound did not show any mutagenic activity in the Salmonella typhimurium tester strains TA1535, TA100, TA1537, TA1538, TA98 in the presence and absence of S9 metabolic activation system and hence is not mutagenic in vitro.
In vitro gene mutation assay in mammalian cell
In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 5, 10, 20, 30 , 40 or 60 nL/mL (trial 1), 0, 20, 30, 40, 50, 60 or 80 nL/mL (trial 2), 0, 10, 20, 30, 40, 60 or 80 nL/mL (trial 3) or 0, 10, 20, 30 or 40 nL/mL (trial 4) without S9 and 0, 30, 40, 50, 60, 80 or 100 nL/mL (trial 1) and 0, 30, 40, 50, 60, 80 or 100 nL/mL (trial 2) with S9. Mouse lymphoma L5178Y cells were maintained at 37˚C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine-resistant cells, subcultures were exposed to medium containing THMG (thymidine, hypoxanthine, methotrexate, and glycine) for 1 day, to medium containing THG (thymidine, hypoxanthine, and glycine) for 1 day, and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 106cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with butyl benzyl phthalate continued for 4 hours, at which time the medium plus butyl benzyl phthalate was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 106cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT-resistant (TK-1) cells; 600 cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37" C in 5% CO2 for 10 to 12 days. The test was initially performed without 59.' If a clearly positive response was not obtained, the test was repeated using freshly prepared S9 from the livers of Aroclor 1254 induced maleFischer344/N rats. Based on the observations made, thetest chemical did not induce gene mutation inMouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.
In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 1.25, 2.5, 3.75, 5.0 or 7.5µg/mL (trial 1), 0, 2, 3, 4, 5, 6 or 8µg/mL (trial 2), 0, 2, 3, 4, 5, 6, 8 or 10µg/mL (trial 3) without S9 and 0, 2.5, 5.0, 7.5, 10 or 15µg/mL (trial 1) with S9. Mouse lymphoma L5178Y cells were maintained at 37˚C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine-resistant cells, subcultures were exposed to medium containing THMG (thymidine, hypoxanthine, methotrexate, and glycine) for 1 day, to medium containing THG (thymidine, hypoxanthine, and glycine) for 1 day, and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 106cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with butyl benzyl phthalate continued for 4 hours, at which time the medium plus butyl benzyl phthalate was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 106cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT-resistant (TK-1) cells; 600 cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37˚C in 5% CO2 for 10 to 12 days. Based on the observations made, thetest chemical did not induce gene mutation in Mouse lymphoma L5178Y cells in the presence S9 metabolic activation system. It however induced gene mutation I the cell line in the absence of S9 metabolic activation system.
Based on the data summarized disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)benzoate (17372-87-1) is expected to not induce gene mutation In vitro. Hence it is not likely to be mutagenic in vitro.
Justification for classification or non-classification
Thus based on the above annotation and CLP criteria for target substance disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)benzoate (17372-87-1) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.
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