Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 231-679-3 | CAS number: 7681-82-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames assay:
Ames assay was performed to investigate the potential of the test chemical 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 theSalmonella typhimuriumstrains 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 and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment.
Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.050, 0.158, 0.501, 1.582 and 5 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 with the test chemical (CAS no 7681-82-5) 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 and positive controls are 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 chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.
In vitro mammalian micronucleus assay:
The test chemical did not induce any increase in the frequency of MNBN cells for doses ranging from 0.625 to 10 mM in the micronucleus assay in the CHO cell line 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
- 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 the test chemical 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
- 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 (NC), 0.050, 0.158, 0.501, 1.582 and 5 mg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The test chemical was solulble in Distilled water - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Distilled 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 data
- 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 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 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 below for the Trial-I (Plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or 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.
In TA 98 and TA 100 there was no reduction in colony count as well as in background lawn was observed in treated concentration 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: 0.0 (NC), 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).
The concentrations used in the experiment (pre-experiment, Trial-1, Trial-2) were placed with (√10) half log interval.
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:
- The test chemical 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 the test chemical 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 and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment.
Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.050, 0.158, 0.501, 1.582 and 5 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 with the test chemical (CAS no 7681-82-5) 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 and positive controls are 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 chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- 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 based on 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 for the target CAS is summarized based on data from various test chemicals
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- No data
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Remarks:
- 1 / 2
- Details on mammalian cell type (if applicable):
- - Type and identity of media: The cell line was grown at 37 Deg C in a humidified atmosphere at 5% CO2 in air, in HAM’S F12 medium with L-glutamine supplemented with 10% fetal calf serum (FCS), penicillin (50 UI/ml) and streptomycine (50 µg/ml). Cells were subcultured 24 h before treatment.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data - Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- No data
- Metabolic activation:
- not specified
- Metabolic activation system:
- no data
- Test concentrations with justification for top dose:
- 1/2. 0.625, 1.25, 2.5, 5 and 10 mM
- Vehicle / solvent:
- 1/2. - Vehicle(s)/solvent(s) used: Culture medium
- Justification for choice of solvent/vehicle: The test chemical was soluble in culture medium - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- not specified
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- MMS (30 µg/ml) / 1/2
- Details on test system and experimental conditions:
- 1/2. METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 150000 cells/well
DURATION
- Preincubation period: No data
- Exposure duration: 3 hrs
- Expression time (cells in growth medium): 20 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): Acridine orange
NUMBER OF REPLICATIONS: Duplicate
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were fixed with cold methanol, stained with acridine orange (62.5 mg/ml) for 5 min and mounted in Sorensen buffer. Slides were coded and blindly examined under an epifluorescence microscope at 1000X magnification under oil immersion.
NUMBER OF CELLS EVALUATED: One thousand (1000) binucleated cells were scored
for each slide.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: MN should be morphologically identical to but smaller than nuclei, their diameter usually varied between 1/6th and 1/3rd of the mean diameter of the main nuclei. MN should be readily distinguished and not be linked to the main nuclei via nucleoplasmic bridges. Cells showing chromatin condensation or nuclear fragmentation with an intact cytoplasmic membrane were classified as apoptotic cells.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, Cytotoxicity was measured by the Binucleate cell ratio between treated and control slides
- 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:
- 1/2. MN should be morphologically identical to but smaller than nuclei, their diameter usually varied between 1/6th and 1/3rd of the mean diameter of the main nuclei. MN should be readily distinguished and not be linked to the main nuclei via nucleoplasmic bridges. Cells showing chromatin condensation or nuclear fragmentation with an intact cytoplasmic membrane were classified as apoptotic cells.
- Statistics:
- 1/2. In the cytokinesis-block micronucleus assay, data were expressed as the percentage of binucleated cells with micronuclei. Comparisons between control and treated cell cultures were made using ANOVA and Dunnett’s one sided test.
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- not specified
- Genotoxicity:
- negative
- Remarks:
- The test chemical did not induce any increase in the frequency of MNBN cells for doses ranging from 0.625 to 10 mM.
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- 1/2. 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
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: In preliminary cytotoxicity assays, CHO cells were
exposed for 1 h to the test compounds at concentrations ranging from 0.001 to 5 mg/ml.
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:
- The test chemical did not induce any increase in the frequency of MNBN cells for doses ranging from 0.625 to 10 mM in the micronucleus assay in the CHO cell line 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 target chemical. The studies are as mentioned below:
Genotoxic effects of the test chemical was evaluated in vitro using the cytokinesis-block micronucleus assay on CHO cells. The study was performed using CHO cells. The test chemical was dissolved in culture medum and used at dose level of 0.625, 1.25, 2.5, 5 and 10 mM. In preliminary cytotoxicity assays, CHO cells were exposed for 1 h to the test compound at concentrations ranging from 0.001 to 5 mg/ml. The cells were exposed for 3 h to concentrations of the test compounds of 0.625, 1.25, 2.5, 5 and 10 mM. Test compound and MMS (30 mg/ml) were dissolved the culture medium. Exponentially growing CHO-K1 cells were plated in a six-well plate on glass coverslips (1.5 X 105 cells/well) and cultured 24 h prior to compound treatment. Duplicate coverslips were established for each experiment, and at least two independent experiments were performed. The cells were exposed to the chemicals at different concentrations for 3 h in a FCS free medium. At the end of treatment, cells were washed twice with PBS before a 20 h incubation in fresh medium containing 10% of FCS and 3 mg/ml of cytochalasin B. Thereafter, cells were washed twice with PBS and allowed to recover for 1.5 h in 10% FCS fresh medium. Cells were fixed with cold methanol, stained with acridine orange (62.5 mg/ml) for 5 min and mounted in Sorensen buffer. Slides were coded and blindly examined under an epifluorescence microscope at 1000X magnification under oil immersion. Briefly, the cells should be binucleated (BN) with an intact nuclear membrane and should be situated within the same cytoplasmic boundary. MN should be morphologically identical to but smaller than nuclei, their diameter usually varied between 1/6th and 1/3rd of the mean diameter of the main nuclei. MN should be readily distinguished and not be linked to the main nuclei via nucleoplasmic bridges. Cells showing chromatin condensation or nuclear fragmentation with an intact cytoplasmic membrane were classified as apoptotic cells. One thousand (1000) binucleated cells were scored for each slide. The frequencies of BN, of BN with MN (MNBN) and of apoptotic cells (AP) were estimated. MMS (30 mg/ml), a well known alkylating agent was used as positive control. Cytotoxicity was measured by the BN cell ratio between treated and control slides.
Based on the observations made, the test chemical did not induce any increase in the frequency of MNBN cells for doses ranging from 0.625 to 10 mM in the micronucleus assay in the CHO cell line and hence it is not likely to classify as a gene mutant in vitro.
Referenceopen allclose all
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 |
122 |
25 |
124 |
25 |
R2 |
116 |
22 |
126 |
26 |
|
R3 |
124 |
23 |
120 |
24 |
|
T1 (0.002) |
R1 |
98 |
15 |
108 |
18 |
R2 |
118 |
17 |
102 |
18 |
|
R3 |
102 |
14 |
104 |
16 |
|
T2 (0.005) |
R1 |
104 |
16 |
108 |
22 |
R2 |
102 |
15 |
110 |
20 |
|
R3 |
106 |
15 |
106 |
18 |
|
T3 (0.016) |
R1 |
104 |
20 |
112 |
20 |
R2 |
110 |
18 |
108 |
18 |
|
R3 |
108 |
20 |
110 |
22 |
|
T4 (0.050) |
R1 |
112 |
17 |
112 |
21 |
R2 |
120 |
20 |
116 |
23 |
|
R3 |
114 |
22 |
110 |
20 |
|
T5 (0.158) |
R1 |
118 |
22 |
120 |
21 |
R2 |
110 |
20 |
122 |
23 |
|
R3 |
106 |
23 |
118 |
22 |
|
T6 (0.501) |
R1 |
110 |
22 |
112 |
24 |
R2 |
118 |
16 |
119 |
22 |
|
R3 |
112 |
17 |
122 |
21 |
|
T7 (1.582) |
R1 |
118 |
22 |
120 |
23 |
R2 |
110 |
23 |
122 |
22 |
|
R3 |
122 |
19 |
118 |
20 |
|
T8 (5) |
R1 |
120 |
22 |
120 |
23 |
R2 |
118 |
23 |
122 |
24 |
|
R3 |
123 |
22 |
124 |
24 |
|
PC |
R1 |
1224 |
1016 |
1488 |
1224 |
R2 |
1240 |
984 |
1456 |
1176 |
|
R3 |
1256 |
992 |
1440 |
1160 |
|
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 |
14 |
25 |
124 |
255 |
R2 |
8 |
13 |
26 |
126 |
246 |
|
R3 |
7 |
15 |
24 |
120 |
252 |
|
T1 (0.050) |
R1 |
5 |
11 |
21 |
112 |
246 |
R2 |
4 |
12 |
23 |
116 |
238 |
|
R3 |
7 |
10 |
20 |
110 |
242 |
|
T2 (0.158) |
R1 |
6 |
10 |
21 |
120 |
230 |
R2 |
4 |
12 |
23 |
122 |
250 |
|
R3 |
7 |
13 |
22 |
118 |
222 |
|
T3 (0.501) |
R1 |
6 |
10 |
24 |
112 |
238 |
R2 |
4 |
14 |
22 |
119 |
248 |
|
R3 |
5 |
9 |
21 |
122 |
229 |
|
T4 (1.582) |
R1 |
7 |
10 |
23 |
120 |
240 |
R2 |
7 |
11 |
22 |
122 |
248 |
|
R3 |
5 |
13 |
20 |
118 |
250 |
|
T5 (5) |
R1 |
6 |
12 |
23 |
120 |
232 |
R2 |
7 |
12 |
24 |
122 |
243 |
|
R3 |
5 |
14 |
24 |
124 |
244 |
|
PC |
R1 |
186 |
548 |
1224 |
1488 |
1280 |
R2 |
190 |
480 |
1176 |
1456 |
1360 |
|
R3 |
171 |
458 |
1160 |
1440 |
1296 |
|
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 |
122 |
253 |
R2 |
7 |
15 |
22 |
116 |
268 |
|
R3 |
7 |
16 |
23 |
124 |
276 |
|
T1 (0.050) |
R1 |
6 |
14 |
17 |
112 |
256 |
R2 |
4 |
10 |
20 |
120 |
255 |
|
R3 |
5 |
9 |
22 |
114 |
240 |
|
T2 (0.158) |
R1 |
5 |
15 |
22 |
118 |
220 |
R2 |
6 |
12 |
20 |
110 |
232 |
|
R3 |
5 |
10 |
23 |
106 |
248 |
|
T3 (0.501) |
R1 |
6 |
12 |
22 |
110 |
242 |
R2 |
6 |
12 |
16 |
118 |
244 |
|
R3 |
5 |
11 |
17 |
112 |
250 |
|
T4 (1.582) |
R1 |
6 |
15 |
22 |
118 |
242 |
R2 |
4 |
10 |
23 |
110 |
258 |
|
R3 |
6 |
14 |
19 |
122 |
260 |
|
T5 (5) |
R1 |
7 |
15 |
22 |
120 |
266 |
R2 |
5 |
14 |
23 |
118 |
250 |
|
R3 |
6 |
13 |
22 |
123 |
246 |
|
PC |
R1 |
168 |
1240 |
1016 |
1224 |
1888 |
R2 |
181 |
1192 |
984 |
1240 |
1664 |
|
R3 |
166 |
1144 |
992 |
1256 |
1608 |
|
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 |
7 |
16 |
23 |
121 |
273 |
R2 |
6 |
15 |
25 |
119 |
253 |
|
R3 |
8 |
15 |
26 |
118 |
260 |
|
T1 (0.050) |
R1 |
5 |
9 |
21 |
102 |
238 |
R2 |
6 |
12 |
19 |
105 |
226 |
|
R3 |
6 |
12 |
20 |
103 |
220 |
|
T2 (0.158) |
R1 |
6 |
13 |
22 |
109 |
226 |
R2 |
5 |
12 |
25 |
110 |
242 |
|
R3 |
5 |
13 |
23 |
114 |
240 |
|
T3 (0.501) |
R1 |
6 |
15 |
25 |
115 |
248 |
R2 |
4 |
16 |
23 |
116 |
246 |
|
R3 |
5 |
12 |
24 |
112 |
234 |
|
T4 (1.582) |
R1 |
7 |
14 |
20 |
118 |
258 |
R2 |
5 |
11 |
23 |
117 |
255 |
|
R3 |
6 |
14 |
25 |
119 |
270 |
|
T5 (5) |
R1 |
6 |
15 |
26 |
109 |
250 |
R2 |
6 |
15 |
24 |
119 |
260 |
|
R3 |
7 |
12 |
23 |
115 |
262 |
|
PC |
R1 |
171 |
232 |
1336 |
1456 |
1430 |
R2 |
184 |
241 |
1360 |
1528 |
1446 |
|
R3 |
192 |
250 |
1284 |
1504 |
1520 |
|
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 |
17 |
25 |
122 |
255 |
R2 |
7 |
17 |
27 |
121 |
260 |
|
R3 |
7 |
14 |
25 |
116 |
278 |
|
T1 (0.050) |
R1 |
6 |
10 |
22 |
104 |
225 |
R2 |
5 |
11 |
20 |
106 |
220 |
|
R3 |
5 |
13 |
19 |
102 |
218 |
|
T2 (0.158) |
R1 |
6 |
12 |
18 |
113 |
218 |
R2 |
5 |
15 |
22 |
116 |
216 |
|
R3 |
4 |
12 |
22 |
115 |
210 |
|
T3 (0.501) |
R1 |
6 |
13 |
23 |
106 |
220 |
R2 |
6 |
11 |
25 |
101 |
224 |
|
R3 |
5 |
12 |
22 |
117 |
232 |
|
T4 (1.582) |
R1 |
7 |
13 |
22 |
118 |
232 |
R2 |
6 |
14 |
24 |
119 |
235 |
|
R3 |
5 |
13 |
20 |
121 |
240 |
|
T5 (5) |
R1 |
7 |
16 |
24 |
120 |
252 |
R2 |
6 |
16 |
26 |
114 |
250 |
|
R3 |
6 |
12 |
26 |
112 |
246 |
|
PC |
R1 |
184 |
1264 |
896 |
1128 |
1576 |
R2 |
178 |
1232 |
936 |
1086 |
1616 |
|
R3 |
166 |
1192 |
966 |
1170 |
1664 |
|
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) |
7.33 |
0.58 |
14.00 |
1.00 |
25.00 |
1.00 |
123.33 |
3.06 |
251.00 |
4.58 |
T1 (0.050) |
5.33 |
1.53 |
11.00 |
1.00 |
21.33 |
1.53 |
112.67 |
3.06 |
242.00 |
4.00 |
T2 (0.158) |
5.67 |
1.53 |
11.67 |
1.53 |
22.00 |
1.00 |
120.00 |
2.00 |
234.00 |
14.42 |
T3 (0.501) |
5.00 |
1.00 |
11.00 |
2.65 |
22.33 |
1.53 |
117.67 |
5.13 |
238.33 |
9.50 |
T4 (1.582) |
6.33 |
1.15 |
11.33 |
1.53 |
21.67 |
1.53 |
120.00 |
2.00 |
246.00 |
5.29 |
T5 (5) |
6.00 |
1.00 |
12.67 |
1.15 |
23.67 |
0.58 |
122.00 |
2.00 |
239.67 |
6.66 |
PC |
182.33 |
10.02 |
495.33 |
46.92 |
1186.67 |
33.31 |
1461.33 |
24.44 |
1312.00 |
42.33 |
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) |
6.67 |
0.58 |
15.00 |
1.00 |
23.33 |
1.53 |
120.67 |
4.16 |
265.67 |
11.68 |
T1 (0.050) |
5.00 |
1.00 |
11.00 |
2.65 |
19.67 |
2.52 |
115.33 |
4.16 |
250.33 |
8.96 |
T2 (0.158) |
5.33 |
0.58 |
12.33 |
2.52 |
21.67 |
1.53 |
111.33 |
6.11 |
233.33 |
14.05 |
T3 (0.501) |
5.67 |
0.58 |
11.67 |
0.58 |
18.33 |
3.21 |
113.33 |
4.16 |
245.33 |
4.16 |
T4 (1.582) |
5.33 |
1.15 |
13.00 |
2.65 |
21.33 |
2.08 |
116.67 |
6.11 |
253.33 |
9.87 |
T5 (5) |
6.00 |
1.00 |
14.00 |
1.00 |
22.33 |
0.58 |
120.33 |
2.52 |
254.00 |
10.58 |
PC |
171.67 |
8.14 |
1192.00 |
48.00 |
997.33 |
16.65 |
1240.00 |
16.00 |
1720.00 |
148.16 |
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
TABLE 5 - MEAN REVERTANT COUNT IN PRE-INCUBATION METHOD (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) |
7.00 |
1.00 |
15.33 |
0.58 |
24.67 |
1.53 |
119.33 |
1.53 |
262.00 |
10.15 |
T1 (0.050) |
5.67 |
0.58 |
11.00 |
1.73 |
20.00 |
1.00 |
103.33 |
1.53 |
228.00 |
9.17 |
T2 (0.158) |
5.33 |
0.58 |
12.67 |
0.58 |
23.33 |
1.53 |
111.00 |
2.65 |
236.00 |
8.72 |
T3 (0.501) |
5.00 |
1.00 |
14.33 |
2.08 |
24.00 |
1.00 |
114.33 |
2.08 |
242.67 |
7.57 |
T4 (1.582) |
6.00 |
1.00 |
13.00 |
1.73 |
22.67 |
2.52 |
118.00 |
1.00 |
261.00 |
7.94 |
T5 (5) |
6.33 |
0.58 |
14.00 |
1.73 |
24.33 |
1.53 |
114.33 |
5.03 |
257.33 |
6.43 |
PC |
182.33 |
10.60 |
241.00 |
9.00 |
1326.67 |
38.85 |
1496.00 |
36.66 |
1465.33 |
48.01 |
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) |
6.67 |
0.58 |
16.00 |
1.73 |
25.67 |
1.15 |
119.67 |
3.21 |
264.33 |
12.10 |
T1 (0.050) |
5.33 |
0.58 |
11.33 |
1.53 |
20.33 |
1.53 |
104.00 |
2.00 |
221.00 |
3.61 |
T2 (0.158) |
5.00 |
1.00 |
13.00 |
1.73 |
20.67 |
2.31 |
114.67 |
1.53 |
214.67 |
4.16 |
T3 (0.501) |
5.67 |
0.58 |
12.00 |
1.00 |
23.33 |
1.53 |
108.00 |
8.19 |
225.33 |
6.11 |
T4 (1.582) |
6.00 |
1.00 |
13.33 |
0.58 |
22.00 |
2.00 |
119.33 |
1.53 |
235.67 |
4.04 |
T5 (5) |
6.33 |
0.58 |
14.67 |
2.31 |
25.33 |
1.15 |
115.33 |
4.16 |
249.33 |
3.06 |
PC |
176.00 |
9.17 |
1229.33 |
36.07 |
932.67 |
35.12 |
1128.00 |
42.00 |
1618.67 |
44.06 |
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 available for the target chemical and its read across chemical was reviewed to determine the mutagenic nature. The chromosome aberration study for the target chemical is currently ongoing and would be updated in the dossier upon reciept of the study reports. The target and read across studies are as mentioned below:
Gene mutation in vitro:
Ames assay:
Ames assay was performed to investigate the potential of the test chemical 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 and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment.
Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.050, 0.158, 0.501, 1.582 and 5 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 with the test chemical (CAS no 7681-82-5) 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 and positive controls are 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 chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.
In vitro mammalian micronucleus assay:
Genotoxic effects of the 2 test chemicals was evaluated in vitro using the cytokinesis-block micronucleus assay on CHO cells. The study was performed using CHO cells. The test chemical was dissolved in culture medum and used at dose level of 0.625, 1.25, 2.5, 5 and 10 mM. In preliminary cytotoxicity assays, CHO cells were exposed for 1 h to the test compound at concentrations ranging from 0.001 to 5 mg/ml. The cells were exposed for 3 h to concentrations of the test compounds of 0.625, 1.25, 2.5, 5 and 10 mM. Test compound and MMS (30 mg/ml) were dissolved the culture medium. Exponentially growing CHO-K1 cells were plated in a six-well plate on glass coverslips (1.5 X 105cells/well) and cultured 24 h prior to compound treatment. Duplicate coverslips were established for each experiment, and at least two independent experiments were performed. The cells were exposed to the chemicals at different concentrations for 3 h in a FCS free medium. At the end of treatment, cells were washed twice with PBS before a 20 h incubation in fresh medium containing 10% of FCS and 3 mg/ml of cytochalasin B. Thereafter, cells were washed twice with PBS and allowed to recover for 1.5 h in 10% FCS fresh medium. Cells were fixed with cold methanol, stained with acridine orange (62.5 mg/ml) for 5 min and mounted in Sorensen buffer. Slides were coded and blindly examined under an epifluorescence microscope at 1000X magnification under oil immersion. Briefly, the cells should be binucleated (BN) with an intact nuclear membrane and should be situated within the same cytoplasmic boundary. MN should be morphologically identical to but smaller than nuclei, their diameter usually varied between 1/6th and 1/3rd of the mean diameter of the main nuclei. MN should be readily distinguished and not be linked to the main nuclei via nucleoplasmic bridges. Cells showing chromatin condensation or nuclear fragmentation with an intact cytoplasmic membrane were classified as apoptotic cells. One thousand (1000) binucleated cells were scored for each slide. The frequencies of BN, of BN with MN (MNBN) and of apoptotic cells (AP) were estimated. MMS (30 mg/ml), a well known alkylating agent was used as positive control. Cytotoxicity was measured by the BN cell ratio between treated and control slides. Based on the observations made, the test chemicals did not induce any increase in the frequency of MNBN cells for doses ranging from 0.625 to 10 mM in the micronucleus assay in the CHO cell line and hence it is not liekly to classify as a gene mutant in vitro.
Based on the data available for the target chemical and its various read across chemicals, the test chemical does not exhibit gene mutation in vitro. Hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Justification for classification or non-classification
Based on the data available for the read across chemical, the test chemical does not enhibit gene mutation in vitro. Hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
