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EC number: 271-178-7 | CAS number: 68516-75-6
- Life Cycle description
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- Endpoint summary
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
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- 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
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- Toxicological Summary
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- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
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- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Two Ames tests one with and one without azo-dye modification (OECD TG 471), a HPRT assay in CHO cells (OECD TG 476) and a chromosome aberration assay in human lymphocytes (OECD TG 473) revealed no mutagenicity or genotoxicity of the test substance, Pigment Brown 41.
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:
- 10 December 2021 to 05 February 2022
- 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)
- Version / remarks:
- 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- s9 Liver homogenate
- Test concentrations with justification for top dose:
- At a concentration of 0.125 mg/mL, the Relative Survival was greater than 10%. Therefore 0.125 mg/mL was selected as the highest concentration for testing in gene mutation test.
- Vehicle / solvent:
- DMSO
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- benzo(a)pyrene
- Details on test system and experimental conditions:
- CHO AA8 Cells, a derivative of the CHO-K1, were used, CHO AA8 cells, Batch No.5000062 procured from American Type Culture Collection (ATCC) was used for the test.
- Evaluation criteria:
- A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
The increase is concentration-related when evaluated with an appropriate trend test.
Any of the results are outside the distribution of the historical negative/vehicle control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
A test chemical is considered clearly negative if, in all experimental conditions examined:
None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
There is no concentration-related increase when evaluated with an appropriate trend test.
All results are inside the distribution of the historical negative/vehicle control data. - Statistics:
- Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups by performing power transformation procedure by Snee and Irr (1981) with which, the observed mutant frequency was transformed using the formula:
Y=(X+A)B
Where,
Y = transformed mutant frequency, X = observed mutant frequency
BIO-GNT 1743 Study Report Page 19 of 36
[Where X=No. of mutant colonies per replicateACE value×100
and A, B = constants (viz. A = 1 and B = 0.15)]
Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0. If the analysis of variance is significant at p < 0.05, Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control.
The statistical significances are designated by the superscripts as given below:
* Statistically significant (p<0.05) change than the vehicle control group. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- 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:
- Solubility, Precipitation and pH Test
The test item was found soluble in DMSO at 200 mg/mL. Precipitation test was conducted at 0.0625, 0.125, 0.25, 0.50, 1 and 2 mg/mL concentrations. Post 3 hours and 40 minutes of incubation, no precipitation observed at 0.125 and 0.0625 mg/mL, mild precipitation was observed at 0.25 mg/mL, moderate precipitation observed at 0.5 mg/mL and heavy precipitation observed at 1 and 2 mg/mL. No change in pH was observed at the tested concentrations up to 2 mg/mL.
Initial Cytotoxicity Test
In the initial cytotoxicity test, there was no evidence of excessive cytotoxicity (˂10% RS) at and up to 0.5 mg/mL in both presence of metabolic activation and absence of metabolic activation when compared to vehicle control. In the presence of metabolic activation, the RS values ranged from 1.23 % to 45.68 % and in the absence of metabolic activation, the RS values ranged from 1.27 % to 46.84 % at the concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL.
Gene Mutation Test
In the gene mutation test, the cells were treated with test item at the concentrations of 0.015625, 0.03125, 0.0625 and 0.125 mg/mL using DMSO as the vehicle in four plate cultures both in the presence of metabolic activation and absence of metabolic activation.
The test item resulted in mutant frequencies of 23.46 to 24.71 per 2×106 cells in the presence of metabolic activation with 24.18 per 2×106 cells in the vehicle control. In the absence of metabolic activation, mutant frequencies of 24.10 to 24.68 per 2×106 cells were observed with 23.86 per 2×106 cells in the vehicle control. There was no statistically significant increase in the mutant frequencies observed when compared with vehicle control at any of the tested concentrations.
There was no evidence of excessive cytotoxicity (˂10% RS) at any of the concentrations both in presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 17.07 to 85.37 % and in the absence of metabolic activation the RS values ranged from 18.99 to 78.48 % respectively.
Application of the positive control, 3 μg/mL of Benzo (a) pyrene, resulted in a RS value of 89.02 % in the presence of metabolic activation, and a mutant frequency of 259.55 per 2×106 cells which was statistically significant when compared with the vehicle control.
The treatment with the positive control, 1 μg/mL of 4-Nitroquinoline N-oxide, resulted in a RS value 96.20 % in the absence of metabolic activation and a mutant frequency of 262.92 per 2×106 cells and was statistically significant when compared with that of vehicle control. - Remarks on result:
- no mutagenic potential (based on QSAR/QSPR prediction)
- Conclusions:
- Based on the results obtained, the test item is considered as non-mutagenic at and up to the concentration of 0.125 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.
- Executive summary:
The test item was evaluated for gene mutation test in CHO AA8 cells.
The test item was found soluble in DMSO at 200 mg/mL. Precipitation test was conducted at 0.0625, 0.125, 0.25, 0.50, 1 and 2 mg/mL concentrations. After 3 hours and 40 minutes of incubation, no precipitation was observed at 0.125 and 0.0625 mg/mL, mild precipitation was observed at 0.25 mg/mL, moderate precipitation was observed at 0.5 mg/mL and heavy precipitation was observed at 1 and 2 mg/mL. No change in pH was observed at the tested concentrations up to 2 mg/mL.
On the basis of precipitation results, 0.5 mg/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 33 minutes). Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test.
The results of the initial cytotoxicity test indicated that the Relative Survival was greater than 10% at 0.125 mg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 0.125 mg/mL was selected as highest concentration for gene mutation test.
The gene mutation test was conducted at the concentrations of 0.015625, 0.03125, 0.0625 and 0.125 mg/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (4 hours).
Benzo(a) pyrene and 4 Nitroquinoline N-oxide were used as Positive controls for the gene mutation test.
Cytotoxicity as Relative Survival was 17.07 to 18.99 % in presence of metabolic activation and absence of metabolic activation at the highest tested concentration of 0.125 mg/mL.
There was no statistically significant increase in mutant frequencies at any of the concentrations tested when compared with the vehicle control. Moreover, treatment with the test item resulted in mutant frequencies which were within acceptable ranges with regard to historical controls.
There was statistically significant increase in mutant frequencies for positive controls when compared with the vehicle control in both metabolic activation and absence of metabolic activation.- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 03 December 2021 to 11 March 2022
- 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)
- Version / remarks:
- 29th July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes:
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Liver Homogenate
- Test concentrations with justification for top dose:
- Initial cytotoxicity was conducted at concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL. The percentage reduction in Mitotic Index was in the range of 34.20 to 39.92 at 0.25 mg/mL. As the percentage reduction in MI was not more than 45±5% at 0.25 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.625 and 0.125 mg/mL.
- Vehicle / solvent:
- Dimethyl Sulphoxide
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- Human Peripheral Blood Lymphocytes
- Evaluation criteria:
- All slides including vehicle control, treatment and positive controls of chromosomal aberration test were coded before evaluation.
Cytotoxicity was determined by calculating percentage reduction in mitotic index (%).
Concurrent measures of mitotic index for all treated and vehicle control cultures were determined.
The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate and the metaphases with aberrations were recorded in raw data.
Gaps were recorded separately and reported but not included in the total aberration frequency.
Individual culture data for chromosomal aberrations is provided - Statistics:
- Data (Percentage of cells with aberrations) was analyzed using SPSS Software version 22 for differences among solvent/vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p<0.05) and the statistical significance was designated by the superscripts through out the report as stated below:
* Statistically significant (p<0.05) change than the vehicle control group. - Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The reduction in MI observed at 0.25 mg/mL was 40.15% in the presence of metabolic activation and 41.85% in the absence of metabolic activation for short term treatments. Similarly, 42.24% in the absence of metabolic activation system for long term treatment.
The observed mean percent aberrated cells at 0.0625, 0.125 and 0.25 mg/mL were 1.00, 1.00 and 1.33 in the presence of metabolic activation (short term treatment 3 to 6 hours), 1.00, 1.00 and 1.33 in the absence of metabolic activation (short term treatment 3 to 6 hours) and 1.33, 1.33 and 1.33 in the absence of metabolic activation (long term 20 to 24 hours) respectively.
There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentration levels tested.
Positive control, 10 µg/mL of Cyclophosphamide Monohydrate, in the presence of metabolic activation (3 to 6 hours), induced 10.00% of aberrated cells which was statistically significant compared to the vehicle control (1.00%). The reduction in mitotic index was 9.97% when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (3 to 6 hours), induced 10.00% of aberrated cells which was statistically significant to the vehicle control (1.00%). The reduction in mitotic index observed was 9.90% when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (20 to 24 hours), induced 10.34% of aberrated cells which was statistically significant to the vehicle control (1.33%). The reduction in mitotic index observed was 10.43% when compared with the vehicle control for long term treatment. - Conclusions:
- Based on the results obtained, the test item is considered as non-clastogenic upto the concentration of 0.25 mg/mL both in the presence and absence of metabolic activation under the presented test conditions.
- Executive summary:
The test item, was evaluated for chromosomal aberrations in human lymphocytes.
The test item formed homogeneous brown solution in DMSO at 200 mg/mL. Precipitation and pH test was conducted at 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 24 hours and 15 minutes of incubation, heavy precipitation was observed at 1 and 2 mg/mL, moderate precipitation was observed at 0.5, mg/mL, mild precipitation was observed at 0.25 mg/mL. No precipitation was observed at 0.125 and 0.625 mg/mL. No change in pH was observed in any of the concentrations tested upto 2 mg/mL. Hence, 0.5 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.03125, 0.0625, 0.125 and 0.25 mg/mL of test item.
In initial cytotoxicity test, the percentage reduction in Mitotic Index was in the range of 57.90 to 60.08% at 0.5mg/mL in presence of metabolic activation (short term), in absence of metabolic activation (short term) in absence of metabolic activation (long term), respectively. The percentage reduction in mitotic index was in the range of 7.90 to 39.92 at 0.03125 to 0.25 mg/mL. As the percentage reduction inmitotic index was not more than 45±5% at 0.25 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.125 and 0.0625 mg/mL.
In the chromosomal aberration test, the cells were treated with test item at the concentrations of 0.0625, 0.125 and 0.25mg/mLusing DMSO as the vehicle. The treatment was carried out in duplicates for the short term period (3 to 6 hours) both in the presence and absence of metabolic activation and for the long term period (20 to 24 hours) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at the concentration of 10 µg/mL and Mitomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls.
The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, thecells were treated with a metaphase-arresting substance (colchicine), stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.
There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the test item concentration levels tested.Positive controls induced statistically significant aberrant cells when compared to vehicle control.
The concurrent vehicle control values were within the 95% control limits of the distribution of the laboratory’s historical vehicle control database.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant study, similar to OECD 471, Very limited information on test item
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- no independent repeat assay
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Species / strain / cell type:
- S. typhimurium, other: TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- induced rat liver S9
- Test concentrations with justification for top dose:
- 10, 50, 100, 500, 1000 and 5000 µg/plate
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-AA with S9; AF2 and sodium azide without S9
- Evaluation criteria:
- "Significant increases"
- 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- 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:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium, other: TA 1538
- 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:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 23 September 2022 to 09 February 2023
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted on 26th June 2020
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- At the histidine locus of Salmonella typhimurium and at the tryptophan locus of Escherichia coli tester strains.
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 homogenate of hamster liver
- Test concentrations with justification for top dose:
- Based on cytotoxicity results 0.1 mg/plate was considered as the highest test concentration for mutation assay along with 0.001, 0.003, 0.01, 0.03 mg/plate.
- Vehicle / solvent:
- Dimethyl sulphoxide
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: 2-Aminoanthracene
- Details on test system and experimental conditions:
- Preincubation method was carried out with concentrations of 0.001, 0.003, 0.01, 0.03 and 0.1 mg/plate of test item, vehicle and positive control. The tester strains along with S9/PBS were transferred into sterile tubes and kept in an incubator shaker for 30 minutes at 180±5 rpm at temperature 37±1°C. Post incubation, the test constituents were transferred into sterile tubes and mixed with 2 mL molten soft agar containing histidine-biotin for Salmonella typhimurium and L-tryptophan for E.coli WP2 uvrA (pKM101) and poured on to MGA plates. Five concentrations of the test item were plated with each of the following tester strains Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (pKM101) with and without metabolic activation. Plates were incubated at 37±1°C for 63 hours and 32 minutes.
The condition of the bacterial background lawn was evaluated for evidence of the test item cytotoxicity using the code system and revertant colonies for each strain within the test item dilution series were counted manually. - Rationale for test conditions:
- The Bacterial reverse mutation test was considered acceptable as it meets the following criteria:
• The positive control substances produced a significant increase in mutant colony frequencies.
• The spontaneous reversion rates in the solvent and negative control (if used) are in the range of in house historical control data and preferably within the range reported in the literature or if it’s a new batch as per certificate of analysis received by the vendor.
• The tester strains must meet all required genetic characterization.
• Tester Strains used should be in the approximate range of 1×109 to 2×109 cells/mL.
• Minimum of five analyzable test item dose concentrations should be used to evaluate the mutation assay and none of these must show any signs of contamination.
• Cytotoxicity must be defined as greater than 50% reduction in mean revertants per concentration relative to the mean solvent/vehicle control value or by a thinning of the bacterial background lawn.
Study met all criteria mentioned above. - Evaluation criteria:
- The conditions necessary for determining a positive result are: There should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the limit dose tested is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and Escherichia coli WP2 uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Based on the above interpretation, the test item is evaluated as negative. - Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Post incubation test item color interference was observed from 5 mg/plate to 0.2 mg/plate which was interfering with lawn evaluation and for counting of revertant colonies. Hence 5 mg/plate to 0.2 mg/plate was not considered further.
- Conclusions:
- Based on the results of the study, it is concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the concentration of 0.1 mg/plate under the test conditions, when tested on Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) tester strains.
- Executive summary:
The test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test.
The test item formed suspension in dimethyl sulphoxide at a concentration of 50 mg/mL and resulted in no precipitation from 0.00625 to 5 mg/plate at tested concentrations.
On the basis of precipitation test, 5 mg/plate was selected as the highest test concentration for initial cytotoxicity test.
Salmonella typhimurium TA100 was exposed to vehicle control 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 5 mg/plate of test item concentrations. Post incubation test item color interference was observed from 5 mg/plate to 0.2 mg/plate which was interfering with lawn evaluation and for counting of revertant colonies. Hence 5 mg/plate to 0.2 mg/plate was not considered for further evaluation
The test item caused no cytotoxicity from 0.00625 to 0.1 mg/plate in the tester strain Salmonella typhimurium TA100 with lawn intensity 4+ (Thick lawn) in the presence and absence of metabolic activation system when compared to vehicle control 4+ (Thick lawn).
Based on the results of initial cytotoxicity test, concentrations of 0.001, 0.003, 0.01, 0.03 and 0.1 mg/plate were selected for testing in the mutation test.
The test item was assessed for its mutagenic effects using Salmonella typhimurium tester strains: TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101). The test item was tested according to the preincubation method in presence and absence of metabolic activation system using DMSO the vehicle and appropriate positive controls were tested simultaneously. Two trials were carried out for this study in triplicate.
Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both the trials in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials. The number of revertant colonies in the positive controls resulted in 3.7 to 15.6 fold increase under comparable conditions.
Referenceopen allclose all
Test Item Concentration (mg/plate) | No. of Revertants/plate | |||||||||||
With S9 | Without S9 | |||||||||||
R1 | R2 | R3 | Average | ±SD | Bacterial Lawn Intensity | R1 | R2 | R3 | Average | ±SD | Bacterial Lawn Intensity | |
Vehicle Control | 110 | 105 | 108 | 108 | 2.5 | *4+,4+,4+ | 96 | 104 | 102 | 101 | 4.2 | *4+,4+,4+ |
0.00625 | 103 | 109 | 106 | 106 | 3.0 | *4+,4+,4+ | 105 | 102 | 101 | 103 | 2.1 | *4+,4+,4+ |
0.0125 | 109 | 104 | 111 | 108 | 3.6 | *4+,4+,4+ | 106 | 101 | 103 | 103 | 2.5 | *4+,4+,4+ |
0.025 | 107 | 105 | 110 | 107 | 2.5 | *4+,4+,4+ | 104 | 100 | 102 | 102 | 2.0 | *4+,4+,4+ |
0.05 | 103 | 105 | 107 | 105 | 2.0 | *4+,4+,4+ | 99 | 104 | 103 | 102 | 2.6 | *4+,4+,4+ |
0.1 | 106 | 108 | 104 | 106 | 2.0 | *4+,4+,4+ | 99 | 101 | 104 | 101 | 2.5 | *4+,4+,4+ |
0.2** | - | - | - | - | - | - | - | - | - | - | - | - |
0.4** | - | - | - | - | - | - | - | - | - | - | - | - |
0.8** | - | - | - | - | - | - | - | - | - | - | - | - |
1.6** | - | - | - | - | - | - | - | - | - | - | - | - |
3.2** | - | - | - | - | - | - | - | - | - | - | - | - |
5** | - | - | - | - | - | - | - | - | - | - | - | - |
Values of Revertants are in Mean±SD
Lawn intensity: 4+= Thick lawn: Distinguished by a healthy (Normal) background lawn compared to vehicle control plates.
*Lawn intensity of replicates 1, 2, 3 respectively.
Note: **Color interference occurred at 0.2 to 5.0 mg/plate. Evaluation of lawn and revertant counts was not possible. These concentrations were excluded from the mutation test.
Preincubation Method
Refer: Appendix - 2
Treatment | Test Concentration (mg/plate) | No. of Revertants (Mean of 3 Plates) | |||||||||||
With S9 | Without S9 | ||||||||||||
Salmonella typhimurium | E. coli WP2 uvrA (pKM 101) | Salmonella typhimurium | E. coli WP2 uvrA (pKM 101) | ||||||||||
TA 98 | TA 100 | TA 1535 | TA 1537 | TA 98 | TA 100 | TA 1535 | TA 1537 | ||||||
Vehicle Control | 100 µL of DMSO | Mean | 32.0 | 108.7 | 22.0 | 9.7 | 78.7 | 28.7 | 105.3 | 17.3 | 7.3 | 72.7 | |
±SD | 3.0 | 3.1 | 2.0 | 2.1 | 3.5 | 1.5 | 1.5 | 1.5 | 1.5 | 3.5 | |||
Lawn Intensity | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ | |||
Test item | 0.001 | Mean | 31.7 | 109.3 | 22.3 | 11.0 | 78.0 | 28.3 | 101.7 | 18.0 | 8.7 | 71.3 | |
±SD | 3.1 | 3.1 | 1.5 | 1.0 | 4.6 | 1.5 | 3.1 | 2.0 | 1.5 | 3.5 | |||
Fold Increase | 1.0 | 1.0 | 1.0 | 1.1 | 1.0 | 1.0 | 1.0 | 1.0 | 1.2 | 1.0 | |||
Lawn Intensity | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ | |||
0.003 | Mean | 32.3 | 109.0 | 23.3 | 10.0 | 77.7 | 28.0 | 102.7 | 18.3 | 9.0 | 72.0 | ||
±SD | 3.1 | 2.0 | 1.5 | 2.0 | 4.2 | 2.0 | 3.1 | 1.5 | 1.0 | 4.6 | |||
Fold Increase | 1.0 | 1.0 | 1.1 | 1.0 | 1.0 | 1.0 | 1.0 | 1.1 | 1.2 | 1.0 | |||
Lawn Intensity | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ | |||
0.01 | Mean | 29.0 | 108.0 | 22.7 | 9.3 | 76.7 | 27.7 | 105.7 | 18.0 | 8.3 | 72.3 | ||
±SD | 3.6 | 3.0 | 2.1 | 2.1 | 3.5 | 1.5 | 4.0 | 2.0 | 1.5 | 2.5 | |||
Fold Increase | 0.9 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.1 | 1.0 | |||
Lawn Intensity | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ | |||
0.03 | Mean | 32.3 | 109.3 | 22.3 | 9.0 | 76.3 | 28.7 | 101.7 | 18.0 | 9.3 | 71.3 | ||
±SD | 3.5 | 3.1 | 2.1 | 1.0 | 2.5 | 1.5 | 2.5 | 1.0 | 1.2 | 4.5 | |||
Fold Increase | 1.0 | 1.0 | 1.0 | 0.9 | 1.0 | 1.0 | 1.0 | 1.0 | 1.3 | 1.0 | |||
Lawn Intensity | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ | |||
0.1 | Mean | 32.3 | 107.0 | 23.3 | 10.0 | 76.0 | 27.3 | 102.0 | 18.0 | 7.7 | 71.7 | ||
±SD | 3.2 | 2.0 | 1.5 | 2.0 | 4.4 | 1.5 | 3.6 | 2.0 | 1.5 | 3.1 | |||
Fold Increase | 1.0 | 1.0 | 1.1 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | |||
Lawn Intensity | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ | |||
Positive Control | 100 µL of respective Positive Control | Mean | 384.0 | 405.7 | 140.3 | 115.3 | 376.3 | 378.3 | 391.0 | 138.3 | 114.7 | 372.0 | |
±SD | 6.6 | 9.3 | 3.8 | 9.5 | 8.7 | 6.5 | 9.0 | 3.1 | 6.0 | 9.5 | |||
Fold Increase | 12.0 | 3.7 | 6.4 | 11.9 | 4.8 | 13.2 | 3.7 | 8.0 | 15.6 | 5.1 | |||
Lawn Intensity | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+,4+ | 4+,4+, 4+ |
Lawn intensity: 4+= Thick lawn: Distinguished by a healthy (Normal) background lawn compared to vehicle control plates.
Values of Revertants are in Mean±SD
Positive controls:
With S9:
For Salmonella typhimurium TA98, TA100, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene
For E.coli uvrA pKM 101 = 30 µg/plate of 2-Aminoanthracene
Without S9:
For TA98: 2 µg/plate of 2-Nitrofluorene
For TA100 and TA1535: 1 µg/plate of Sodium azide
For TA1537: 50 µg/plate of 9-Aminoacridine
For E.coli uvrA pKM 101: 5 µg/plate of 4 Nitroquinoline N-oxide
Treatment | Test Concentration (mg/plate) | Plate No. | No. of Revertants/Plate in triplicates | ||||||||||
With S9 |
| Without S9 | |||||||||||
Salmonella typhimurium | E. coli WP2 uvrA (pKM 101) |
| Salmonella typhimurium | E. coli WP2 uvrA (pKM 101) | |||||||||
TA 98 | TA 100 | TA 1535 | TA 1537 | TA 98 | TA 100 | TA 1535 | TA 1537 | ||||||
Vehicle Control | 100 µL of DMSO | 1 | 32 | 108 | 24 | 12 | 82 |
| 27 | 104 | 17 | 7 | 69 |
2 | 29 | 112 | 20 | 9 | 75 |
| 30 | 107 | 19 | 9 | 73 | ||
3 | 35 | 106 | 22 | 8 | 79 |
| 29 | 105 | 16 | 6 | 76 | ||
Lawn Intensity | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ |
| *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | ||
Test item | 0.001 | 1 | 31 | 110 | 21 | 12 | 83 |
| 27 | 99 | 16 | 7 | 71 |
2 | 35 | 106 | 24 | 10 | 74 |
| 30 | 105 | 18 | 10 | 75 | ||
3 | 29 | 112 | 22 | 11 | 77 |
| 28 | 101 | 20 | 9 | 68 | ||
Lawn Intensity | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ |
| *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | ||
0.003 | 1 | 33 | 111 | 23 | 10 | 79 |
| 28 | 102 | 20 | 8 | 67 | |
2 | 29 | 107 | 25 | 8 | 81 |
| 30 | 106 | 17 | 10 | 76 | ||
3 | 35 | 109 | 22 | 12 | 73 |
| 26 | 100 | 18 | 9 | 73 | ||
Lawn Intensity | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ |
| *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | ||
0.01 | 1 | 26 | 111 | 25 | 10 | 77 |
| 28 | 110 | 20 | 8 | 75 | |
2 | 28 | 105 | 22 | 7 | 73 |
| 29 | 102 | 18 | 10 | 70 | ||
3 | 33 | 108 | 21 | 11 | 80 |
| 26 | 105 | 16 | 7 | 72 | ||
Lawn Intensity | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ |
| *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | ||
0.03 | 1 | 36 | 110 | 24 | 9 | 74 |
| 29 | 99 | 18 | 10 | 71 | |
2 | 32 | 106 | 23 | 8 | 79 |
| 27 | 102 | 17 | 8 | 76 | ||
3 | 29 | 112 | 20 | 10 | 76 |
| 30 | 104 | 19 | 10 | 67 | ||
Lawn Intensity | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ |
| *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | ||
0.1 | 1 | 36 | 109 | 22 | 12 | 73 |
| 26 | 98 | 20 | 6 | 69 | |
2 | 30 | 105 | 23 | 10 | 81 |
| 29 | 105 | 16 | 9 | 75 | ||
3 | 31 | 107 | 25 | 8 | 74 |
| 27 | 103 | 18 | 8 | 71 | ||
Lawn Intensity | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ |
| *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | ||
Positive Control | 100 µL of respective Positive Control | 1 | 391 | 410 | 142 | 125 | 386 |
| 385 | 400 | 141 | 109 | 363 |
2 | 378 | 412 | 136 | 106 | 374 |
| 372 | 382 | 135 | 114 | 371 | ||
3 | 383 | 395 | 143 | 115 | 369 |
| 378 | 391 | 139 | 121 | 382 | ||
Lawn Intensity | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ |
| *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ | *4+, 4+,4+ |
Lawn intensity: 4+= Thick lawn: Distinguished by a healthy (Normal) background lawn compared to vehicle control plates.
*Lawn intensity of replicates 1, 2, 3 respectively.
Required CFU/mL | No. of colonies of 10-7 dilutions in triplicates | Average | Obtained CFU/mL (1 to 2 × 109 CFU /mL) | |||
R1 | R2 | R3 | ||||
Salmonella typhimurium TA98 | 1-2×109 | 175 | 171 | 168 | 171 | 1.71×109 |
Salmonella typhimurium TA100 | 1-2×109 | 174 | 165 | 180 | 173 | 1.73×109 |
Salmonella typhimurium TA1535 | 1-2×109 | 161 | 179 | 185 | 175 | 1.75×109 |
Salmonella typhimurium TA1537 | 1-2×109 | 182 | 170 | 165 | 172 | 1.72×109 |
E.coli Wp2 uvrA pKM 101 | 1-2×109 | 172 | 181 | 168 | 174 | 1.74×109 |
R: Replicate
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the third time in Directive EC 618/2012.
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.
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