Registration Dossier
Registration Dossier
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EC number: 944-092-9 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
No genotoxic effect was seen in the bacterial reverse mutation test and the gene mutation test in mammalial cells
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 1991-03-27 to 1991-04-12
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The test substance caused no reduction in the growth of the bacterial lawn at any dose level either with or without metabolic activation. Kayarus Supra Rubine BLN was therefore tested up to the maximum recommended dose level of 5000 µg/plate.
- Conclusions:
- No significant increase in the frequency of revertant colonies was recorded for any bacterial strain used in two separate experiments either with or without metabolic activation.
- Executive summary:
A genetic toxicity test was performed to assess the mutagenic potential of the test substance using a bacterial/microsome test system. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA 100, TA100, TA 1535 and TA 1537) and the trytophan-requiring auxotroph strains of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from rat liver.
Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems, not available in the bacterial cell, the test material and the bacteria were also incubated in the presence of a liver microsomal fraction (S9) obtained from rats previously treated with a compound known to induce and elevated level of these enzyme.
The test study included a:
1) Preliminary toxicity study
The dose range used in the preliminary toxicity study was 0, 312.5, 625, 1250, 2500 and 5000 µg/plate. The test material was non-toxic in the strains of bacteria used (TA100 and WP2uvrA).
2) Mutation study
The results for the checks on characteristics, viability spontaneous reversion rate for each tester strain were all found to be satisfactory.
No toxicity was exhibited to any of the strains of bacteria used.
No significant increase in the frequency of revertant colonies was recorded for any bacterial strain used in two separate experiments either with or without metabolic activation. An appropriate allowance of the purity of the test substance was made when the test material solution were prepared. A statistical analysis of the data was not required for the evaluation of the result.
The positive control substance all produced marked increased in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.
Therefore the test substance was found to be non-mutagenic under the conditions of this test
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- read-across source
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Strain: Chinese hamster ovary cells (CHO-K1, BCRC 60006)
- Source: Food Industry Research and Development Institute
- Culture condition: Used Ham's F-12 medium (including 2.0 mM L-Glutamine) with 10% fetal bovine serum (FBS) at an adjusted pH value range from 7.2 to 7.4 and incubated at an 37 ± 1°C incubator with 5 ± 1% CO2 (v/v). - Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver microsomal fraction S9 mix
- Test concentrations with justification for top dose:
- - Main test: with and without S9 mix: 250, 500, 1000, 2000 μg/ml.
- Toxicity data, pre-experiment: 250, 500, 1000, 2000 μg/ml.
- Selection for main test: The highest dosage of the test article is 2.0 mg/mL or according to cell viability test and other test groups are in serial two-fold dilution for three dosages. .
- Spacing factor: the individual concentrations were spaced by a factor of 2. - Vehicle / solvent:
- - Vehicle: culture medium.
- Concentration: NA.
- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
- Formulation preparation: all formulations were prepared freshly before treatment and used within two hours of preparation. - 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:
- Control article:
Negative control:
- Article name: Ham's F-12
- Lot No.: SLBL9236V
- Form: Powder
- Storage condition: Refrigeration (5 ± 3°C)
- Expiration date: 2017/10/31
Positive control (treated with S9 Mix):
- Article name: Benzo [a] pyrene (B [a] P)
- Manufacturer: Sigma-Aldrich
- Lot No.: LC04528V
- Form: Powder
- Storage condition: Room temperature
- Expiration date: 2016/11/30
Positive control (not treated with S9 Mix):
- Article name: 4-Nitroquinoline-l-oxide (4-NQO)
- Manufacturer: Sigma-Aldrich
- Lot No.: WXBB6231V
- Form: Powder
- Storage condition: Freezing (-20 ± 3°C)
- Expiration date: 2019/04/01
Gene mutation test:
- This test was using 10 cm culture dishes to which 10 mL medium was added. Each dish was seeded with 5E5 cells, and then incubated overnight at 37 ± 1°C in the 5 ± 1% CO2 incubator. After incubation, medium was exchanged with 10 mL test article/medium mixture, negative control preparation and positive control items. . After treatment for 3 hours, cells were put in new medium, and then incubated 7 - 9 days at 37 ± 1°C in the 5 ± 1% CO2 incubator.
- After incubation for 7 - 9 days, cells were divided into two groups, one was the colonies formation test group; another was the mutation frequency group. All groups were tested in five copies.
- Colonies formation frequency: 200 cells were seeded in 10 cm dish and incubated 7 days at 37 ± 1°C in the 5 ± 1% CO2 incubator. After incubation, each dish was fixed and dyed to count colonies and calculated the colonies formation frequency.
- Mutation frequency: 1.0 x 106 cells were seeded in 10 cm dish and five copies were used at 37 ± 1°C in the 5 ± 1% CO2 incubator. After cell attachment, 6-thioguanine was added and the final concentration was 10 µg/mL. After incubation for 7 - 12 days at 37 ± 1°C in the 5 ± 1% CO2 incubator, each dish was fixed and dyed to count colonies and the mutation frequency calculated. - Evaluation criteria:
- Criteria for validity and mutagenicity - statistical analysis:
- if the mutation frequency in the positive control group was significantly different from the negative control group (p < 0.05) and the negative control group mutation frequency was spontaneously mutation frequency (5 - 20 x 10-6), the results for this test were considered valid.
- if the mutation frequency in the test group was significantly different from the negative control group (p < 0.05), or showed the dose-related increasing at least in three concentrations, the results were considered positive. - Statistics:
- Organization for Economic Co-operation and Development (OECD). Statistical Issues Related To OECD in vitro Gene Mutation Tests Test Guidelines (TG 476), 2015. France.
The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. - Species / strain:
- Chinese hamster Ovary (CHO)
- 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
- Positive controls validity:
- valid
- Additional information on results:
- Cell viability test:
The cell viability for each test group after treating with or without S9 Mix was analyzed. At the test item concentration of 2.0 mg/mL with or without S9 Mix for 3 hours, the cell viability was greater than 50%. Therefore, 2.0 mg/mL was used as the maximum dosage for the gene mutation test and the number of seeding did not need to be increased.
8.2 Gene mutation test:
The colonies forming frequency and mutation frequency for each test group after treating or not treating with S9 Mix were analyzed.
̶ After incubation with S9 Mix for 3 hours, the negative control group mutation rate was 5.0 x 10-6, which was the spontaneous mutation rate. The positive control group (Benzo [a] pyrene) mutation rate was 247.6 x 10-6, that was significantly different from the negative control group (p < 0.05). At test article concentrations of 2.0 mg/mL, 1.0 mg/mL, 0.5 mg/mL and 0.25 mg/mL, the mutation rates were 4.7 x 10-6, 8.9 x 10-6, 6.4 x 10-6 and 6.1 x 10-6.
̶ After incubation without S9 Mix for 3 hours, the negative control group mutation rate was 29.0 x 10-6, which was the spontaneous mutation rate. The positive control group (4-Nitroquinoline-1-oxide) mutation rate was 116.1 x 10-6, that was significantly different from the negative control group (p < 0.05). At test article concentrations of 2.0 mg/mL, 1.0 mg/mL, 0.5 mg/mL and 0.25 mg/mL the mutation rates were 43.9 x 10-6, 33.5 x 10-6, 14.5 x 10-6 and 17.5 x 10-6.
̶ At statistical analysis, the mutation frequency in each concentration of the test article for 3 hours treatment with or without S9 Mix was not significantly different from the negative control group. - Conclusions:
- The substance is considered to be non-mutagenic in the HPRT assay perfromed.
- Executive summary:
The in vitro mammalian cell gene mutation test was performed in accordance with OECD 476: 2015. and SuperLab SOPF-240 in order to evaluate the test substance for its cytotoxicity and mutation frequency. This test used Chinese hamster ovary cells (CHO-K1). The cytotoxicity determination of the test article for CHO-K1 was used to set the maximum dosage and number of seeding cells in the gene mutation test. In the gene mutation test, test substance concentrations of 250, 500, 1000, and 2000 µg/ml were incubated for 3 hours in the presence or absence of rat liver S9 Mix. The test article maximum concentration of 2.0 mg/mL was determined in a cell viability test where all survival rates for this concentration were greater than 50%. By statistical analysis, the mutation frequency in each concentration of the test article for 3 hours treatment with or without S9 Mix was not significantly different from the negative control group. Therefore, under these test conditions of this study, the test substance was negative in the in vitro mammalian cell gene mutation test.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1991-03-27 to 1991-04-12
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine (Salmonella)
Tryptophan (E. coli WP2 uvr A-) - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- The S9 mix was prepared as follows:S9 5.0 mL, 1.65 M KCL 1.0 mL, 0.4 MMgCl2 1.0mL, 0.1 M Glucose-6-phosphate 2.5 mL, 0.1 M NADP 2.0 mL, 0.2 M Sodium Phosphate buffer (pH 7.4) 25.0 mL, sterile distilled water 13.5 mL
- Test concentrations with justification for top dose:
- With and without the addition of a rat liver homogenate metabolising system at 10% in standard co-factors.
Dose range determined in preliminary toxicity assay: 8 – 5000 µg/plate in first experiment
Dose range used in preliminary: 0, 312.5, 625, 1250, 2500, 5000µg/plate
Experiment repeated on separate day using different cultures of back-strains and fresh chemical solutions. 312.5 – 5000 µg/plate
. - Vehicle / solvent:
- The solvent (sterile distilled water) control plates gave counts of revertant colonies within the normal range.sterile distilled water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- (-S9) -0.2 ug/plate For tester strain TA 98 Migrated to IUCLID6: 0.1 mL of 4NQO was added to a test tube containing 2.0 mL of molten, trace histidine or tryptophan supplemented top agar and 0.5 ml of pH 7.4 buffer.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- (-S9) 80 ug/plate for tester strain TA 1537 Migrated to IUCLID6: 0.1 mL of 9AA was added to a test tube containing 2.0 mL of molten, trace histidine or tryptophan supplemented top agar and 0.5 ml of pH 7.4 buffer.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- (-S9). 2ug/plate for tester strain WP2 uvr A-, 3ug/plate for tester strain TA 100 and 5 ug/plate for tester strain TA 1535 Migrated to IUCLID6: 0.1 mL of ENNG was added to a test tube containing 2.0 mL of molten, trace histidine or tryptophan supplemente
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Remarks:
- 0.1 mL of 2AA was added to a test tube containing 2.0 mL of molten, trace histidine or tryptophan supplemented top agar.
- Positive control substance:
- other: 2-aminoanthracene (2-AA)
- Remarks:
- (+S9) at 2ug/plate for tester strain TA 1535 and 20 ug/plate for tester strain WP2uvrA-
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Remarks:
- 0.1 mL of BP was added to a test tube containing 2.0 mL of molten, trace histidine or tryptophan supplemented top agar.
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- (+S9) at 5 ug/plate for tester strains: TA100, Ta 1537 and TA 98
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
In This assay, overnight Sub-cultures of the master slopes were prepared in nutrient broth( oxoid limited) and incubated at 37 °C for 10 hours. These overnight cultures yielded approximately 10 Exp 8-10 Exp9 bacteria per mL.
SELECTION AGENT (mutation assays):
NUMBER OF REPLICATIONS:
3
NUMBER OF CELLS EVALUATED:
DETERMINATION OF CYTOTOXICITY
- Method:relative total growth - Evaluation criteria:
- Criteria for evaluating Results:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or
more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results then a third experiment may be used
to confirm the correct response. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the Intervals of which should be
between 2 and 5 fold and extend to the limits imposed by toxicity or solubility or up to the maximum recommended dose of 5000 ug/plate. In this case the limiting factor was the maximum recommended dose. - Statistics:
- A statistical analysis of the data was not required for the evaluation of the result.
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Kayarus Supra Rubine BLN caused no reduction in the growth of the bacterial lawn at any dose level either with or without metabolic activation. Kayarus Supra Rubine BLN was therefore tested up to the maximum recommended dose level of 5000 µg/plate.
- Conclusions:
- Interpretation of results (migrated information):
negative No significant increase in the frequency of revertant colonies was recorded for any bacterial strain used with any dose of Kayarus Supra Rubin BLN in two separate experiments either with or without metabolic activation.
No significant Increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of Kayarus Supra Rubine BLN, either with or without metabolic activation. Kayarus Supra Rubine BLN was found to be non-mutagenic under the conditions of this test - Executive summary:
A genetic toxicity test was performed ( in the period of 1991 -03 -27 to 1991 -04 -12) according to Safepharm Definitive Protocol number TX 3094 and was designed to assess the mutagenic potential of KAYARUS SUPRA RUBINE BLN using a bacterial/microsome test system. This in vitro study was based on a technique in which a mutagenic activity is assessed by exposing histidine auxotroph of Salmonella thyphimorium to various concentrations of the test material.
This method is conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EEC an USA, APA (TISCA) guidelines.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium ( Salmonella typhimurium TA98, TA 100, TA100, TA 1535 and TA 1537) and the trytophan-requiring auxotroph strains of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from rat liver.
Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems, not available in the bacterial cell, the test material and the bacteria were also incubated in the presence of a liver microsomal fraction (S9) obtained from rats previously treated with a compound known to induce and elevated level of these enzyme.
The test study included a:
1)Preliminary toxicity study
The dose range of KAYARUS SUPRA RUBINE BLN used in the preliminary toxicity study was 0, 312.5, 625, 1250, 2500 and 5000 µg/plate. The test material was non-toxic in the strains of bacteria used (TA100 and WP2uvrA).
2) Mutation study
The results for the checks on characteristics, viability spontaneous reversion rate for each tester strain were all found to be satisfactory.
No toxicity was exhibited to any of the strains of bacteria used.
No significant increase in the frequency of revertant colonies was recorded for any bacterial strain used with any dose of Kayarus Supra Rubine BLN in two separate experiments either with or without metabolic activation. An appropriate allowance of the purity of the test substance (93.7%) was made when the test material solution were prepared. A statistical analysis of the data was not required for the evaluation of the result.
The positive control substance all produced marked increased in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.
Therefore Kayarus Supra Rubine BLN was found to be non-mutagenic under the conditions of this test
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From Juni to December 2016
- 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:
- 2015
- 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)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Strain: Chinese hamster ovary cells (CHO-K1, BCRC 60006)
- Source: Food Industry Research and Development Institute
- Culture condition: Used Ham's F-12 medium (including 2.0 mM L-Glutamine) with 10% fetal bovine serum (FBS) at an adjusted pH value range from 7.2 to 7.4 and incubated at an 37 ± 1°C incubator with 5 ± 1% CO2 (v/v). - Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver microsomal fraction S9 mix
- Test concentrations with justification for top dose:
- - Main test: with and without S9 mix: 250, 500, 1000, 2000 μg/ml.
- Toxicity data, pre-experiment: 250, 500, 1000, 2000 μg/ml.
- Selection for main test: The highest dosage of the test article is 2.0 mg/mL or according to cell viability test and other test groups are in serial two-fold dilution for three dosages. .
- Spacing factor: the individual concentrations were spaced by a factor of 2. - Vehicle / solvent:
- - Vehicle: culture medium.
- Concentration: NA.
- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
- Formulation preparation: all formulations were prepared freshly before treatment and used within two hours of preparation. - 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:
- Control article:
Negative control:
- Article name: Ham's F-12
- Lot No.: SLBL9236V
- Form: Powder
- Storage condition: Refrigeration (5 ± 3°C)
- Expiration date: 2017/10/31
Positive control (treated with S9 Mix):
- Article name: Benzo [a] pyrene (B [a] P)
- Manufacturer: Sigma-Aldrich
- Lot No.: LC04528V
- Form: Powder
- Storage condition: Room temperature
- Expiration date: 2016/11/30
Positive control (not treated with S9 Mix):
- Article name: 4-Nitroquinoline-l-oxide (4-NQO)
- Manufacturer: Sigma-Aldrich
- Lot No.: WXBB6231V
- Form: Powder
- Storage condition: Freezing (-20 ± 3°C)
- Expiration date: 2019/04/01
Gene mutation test:
- This test was using 10 cm culture dishes to which 10 mL medium was added. Each dish was seeded with 5E5 cells, and then incubated overnight at 37 ± 1°C in the 5 ± 1% CO2 incubator. After incubation, medium was exchanged with 10 mL test article/medium mixture, negative control preparation and positive control items. . After treatment for 3 hours, cells were put in new medium, and then incubated 7 - 9 days at 37 ± 1°C in the 5 ± 1% CO2 incubator.
- After incubation for 7 - 9 days, cells were divided into two groups, one was the colonies formation test group; another was the mutation frequency group. All groups were tested in five copies.
- Colonies formation frequency: 200 cells were seeded in 10 cm dish and incubated 7 days at 37 ± 1°C in the 5 ± 1% CO2 incubator. After incubation, each dish was fixed and dyed to count colonies and calculated the colonies formation frequency.
- Mutation frequency: 1.0 x 106 cells were seeded in 10 cm dish and five copies were used at 37 ± 1°C in the 5 ± 1% CO2 incubator. After cell attachment, 6-thioguanine was added and the final concentration was 10 µg/mL. After incubation for 7 - 12 days at 37 ± 1°C in the 5 ± 1% CO2 incubator, each dish was fixed and dyed to count colonies and the mutation frequency calculated. - Evaluation criteria:
- Criteria for validity and mutagenicity - statistical analysis:
- if the mutation frequency in the positive control group was significantly different from the negative control group (p < 0.05) and the negative control group mutation frequency was spontaneously mutation frequency (5 - 20 x 10-6), the results for this test were considered valid.
- if the mutation frequency in the test group was significantly different from the negative control group (p < 0.05), or showed the dose-related increasing at least in three concentrations, the results were considered positive. - Statistics:
- Organization for Economic Co-operation and Development (OECD). Statistical Issues Related To OECD in vitro Gene Mutation Tests Test Guidelines (TG 476), 2015. France.
The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. - Species / strain:
- Chinese hamster Ovary (CHO)
- 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
- Positive controls validity:
- valid
- Additional information on results:
- Cell viability test:
The cell viability for each test group after treating with or without S9 Mix was analyzed. At the test item concentration of 2.0 mg/mL with or without S9 Mix for 3 hours, the cell viability was greater than 50%. Therefore, 2.0 mg/mL was used as the maximum dosage for the gene mutation test and the number of seeding did not need to be increased.
8.2 Gene mutation test:
The colonies forming frequency and mutation frequency for each test group after treating or not treating with S9 Mix were analyzed.
̶ After incubation with S9 Mix for 3 hours, the negative control group mutation rate was 5.0 x 10-6, which was the spontaneous mutation rate. The positive control group (Benzo [a] pyrene) mutation rate was 247.6 x 10-6, that was significantly different from the negative control group (p < 0.05). At test article concentrations of 2.0 mg/mL, 1.0 mg/mL, 0.5 mg/mL and 0.25 mg/mL, the mutation rates were 4.7 x 10-6, 8.9 x 10-6, 6.4 x 10-6 and 6.1 x 10-6.
̶ After incubation without S9 Mix for 3 hours, the negative control group mutation rate was 29.0 x 10-6, which was the spontaneous mutation rate. The positive control group (4-Nitroquinoline-1-oxide) mutation rate was 116.1 x 10-6, that was significantly different from the negative control group (p < 0.05). At test article concentrations of 2.0 mg/mL, 1.0 mg/mL, 0.5 mg/mL and 0.25 mg/mL the mutation rates were 43.9 x 10-6, 33.5 x 10-6, 14.5 x 10-6 and 17.5 x 10-6.
̶ At statistical analysis, the mutation frequency in each concentration of the test article for 3 hours treatment with or without S9 Mix was not significantly different from the negative control group. - Conclusions:
- The substance is considered to be non-mutagenic in the HPRT assay perfromed.
- Executive summary:
The in vitro mammalian cell gene mutation test was performed in accordance with OECD 476: 2015. and SuperLab SOPF-240 in order to evaluate the test substance for its cytotoxicity and mutation frequency. This test used Chinese hamster ovary cells (CHO-K1). The cytotoxicity determination of the test article for CHO-K1 was used to set the maximum dosage and number of seeding cells in the gene mutation test. In the gene mutation test, test substance concentrations of 250, 500, 1000, and 2000 µg/ml were incubated for 3 hours in the presence or absence of rat liver S9 Mix. The test article maximum concentration of 2.0 mg/mL was determined in a cell viability test where all survival rates for this concentration were greater than 50%. By statistical analysis, the mutation frequency in each concentration of the test article for 3 hours treatment with or without S9 Mix was not significantly different from the negative control group. Therefore, under these test conditions of this study, the test substance was negative in the in vitro mammalian cell gene mutation test.
Referenceopen allclose all
Judgement |
Negative
|
|
Reason for judgement and referential Matters |
No significant increase in the frequency of revertant colonies was recorded for any bacterial strain used with any dose of the test substance in two separate experiments either with or without metabolic activation. An appropriate allowance of the purity of the test substance (93.7%) was made when the test material solution were prepared. A statistical analysis of the data was not required for the evaluation of the result. |
|
Test condition for the Plate method |
||
Composition |
Bacterial suspension 0.1 mL |
|
Test substance solution 0.1 mL |
||
Na-phosphate Buffer 0.5 mL |
||
S9 Mix (incase of metabolic activation method) 0.5 mL |
||
Top Agar solution: 2.0 mL |
||
Incubation |
Temperature/Time 37 °C/ 48 hours |
|
KEY TO TABLE OF RESULTS:
1. When bacterial growth is found, the applicable value is marked with an asterisk.
2. The average number of colonies for each concentration is recorded in parenthesis
3. “Number of revertants”: The observed values and average value are shown in order, beginning with the low concentration of the test substance.
4. The following postfixes are used when required:
C: Contaminated
P: precipitate
X: plate unscorable
ENNG= N-ethyl-N-nitro-N-nitrosoguanidine
4NQO= 4-nitroquinoline-N-oxide
9AA= 9-aminoacridine
2AA= 2-Aminoanthracene
BP= Benzo(a) pyrene
TABLE OF TEST RESULTS: EXPERIMENT I NAME OF THE TEST SUBSTANCE: KAYARUS SUPRA RUBINE BLN
|
|||||||||||
Number of revertants (number of colonies/plate) |
|||||||||||
With (+) S9 or Without (-) S9 |
Test substance concentration (ug/plate) |
Base-pair substitution type |
Fragmentg-type |
||||||||
0 |
TA 100 |
TA 1535 |
W2uvrA- |
TA 98 |
TA 1537 |
||||||
- |
72 |
|
15 |
|
13 |
16 |
|
15 |
|
|
|
100 |
(84.3) |
9 |
(11.7) |
19 (15.7) |
17 |
(16.7) |
16 |
(12.7) |
|
||
81 |
|
11 |
|
15 |
17 |
|
9 |
|
|
||
- |
8.0 |
61 |
|
13 |
|
14 |
16 |
|
14 |
|
|
87 |
(83.0) |
16 |
(14.7) |
13(13.7) |
14 |
(15.7) |
9 |
(10.0) |
|
||
101 |
|
15 |
|
14 |
17 |
|
7 |
|
|
||
- |
40 |
84 |
|
5 |
|
15 |
17 |
|
14 |
|
|
79 |
(76.7) |
13 |
(10.3) |
16(15.0) |
15 |
(16.0) |
10 |
(11.3) |
|
||
67 |
|
13 |
|
14 |
16 |
15 |
10 |
|
|
||
- |
200 |
89 |
|
13 |
|
11 |
15 |
|
14 |
|
|
88 |
(85.3) |
15 |
(14.3) |
13(11.3) |
16 |
16(15.7) |
9 |
(12.7) |
|
||
79 |
|
15 |
|
10 |
16 |
|
15 |
|
|
||
- |
1000 |
96 |
|
11 |
|
7 |
18 |
|
11 |
|
|
85 |
(86.9) |
10 |
(10.3) |
14(11.3) |
17 |
(17.3) |
6 |
(8.7) |
|
||
79 |
|
10 |
|
13 |
17 |
|
9 |
|
|
||
- |
5000 |
60 |
|
11 |
|
11 |
15 |
|
12 |
|
|
76 |
(74.3) |
11 |
(10.0) |
13(11.0) |
18 |
(16.0) |
16 |
(13.7) |
|
||
78 |
|
8 |
|
9 |
15 |
|
13 |
|
|
||
|
Name |
ENNG
|
ENNG |
ENNG |
4NQO |
9AA |
|||||
Positive control not requiring S9 mix |
Concentration (ug/plate) |
3.0
|
5.0 |
2.0 |
0.2 |
80.0 |
|||||
- |
Number of colonies/plate |
510 |
|
178 |
|
544 |
|
166 |
|
148 |
|
546 |
(534.7) |
189 |
(193.3) |
520 |
(534.0) |
156(158.3) |
|
152 |
(151.7) |
||
548 |
|
215 |
|
538 |
|
153 |
|
155 |
|
||
With (+) S9 or Without (-) S9 |
Test substance concentration (ug/plate) |
Base-pair substitution type |
Fragmentg-type |
|
|||||||
|
TA 100 |
TA 1535 |
W2uvrA- |
TA 98 |
TA 1537 |
|
|||||
+ |
0 |
131 |
|
14 |
|
19 |
|
17 |
|
12 |
|
123 |
(116.0) |
19 |
(15.7) |
19 |
(20.3) |
18 (17.7) |
|
15 |
(13.7) |
||
94 |
|
14 |
|
23 |
|
18 |
|
14 |
|
||
+ |
8.0 |
98 |
|
16 |
|
15 |
|
19 |
|
14 |
|
94 |
(100.7) |
11 |
(14.0) |
14 |
(14.0) |
22 (19.7) |
|
12 |
(14.0) |
||
110 |
|
15 |
|
13 |
|
18 |
|
16 |
|
||
+ |
40 |
106 |
|
11 |
|
12 |
|
19 |
|
16 |
|
99 |
(95.3) |
12 |
(10.3) |
9 |
(10.3) |
16 (16.3) |
|
14 |
(15.0) |
||
81 |
|
8 |
|
10 |
|
14 |
|
15 |
|
||
+ |
200 |
71 |
|
13 |
|
18 |
|
16 |
|
13 |
|
110 |
(85.3) |
7 |
(10.3) |
15 |
(17.3) |
15(17.3) |
|
10 |
(13.0) |
||
75 |
|
11 |
|
19 |
|
21 |
|
16 |
|
||
+ |
1000
|
73 |
|
16 |
|
7 |
|
12 |
|
10 |
|
108 |
(97.7) |
17 |
|
14 |
|
17 (14.7) |
|
11 |
(11) |
||
112 |
|
13 |
(15.3) |
15 |
(12.0) |
15 |
|
12 |
|
||
+ |
5000 |
85 |
|
8 |
|
13 |
|
14 |
|
9 |
|
71 |
(75.3) |
8 |
(7.3) |
21 |
(15.7) |
14(14.3) |
|
9 |
(8.7) |
||
70 |
|
7 |
|
13 |
|
15 |
|
8 |
|
||
|
Name |
BP
|
2AA
|
2AA |
BP
|
BP |
|||||
Positive control not requiring S9 mix |
Concentration (ug/plate) |
5.0
|
|
2.0 |
|
20.0 |
|
5.0 |
|
5.0 |
|
|
Number of colonies/plate |
339 |
|
206 |
|
505 |
|
326 |
|
123 |
|
|
|
343 |
(353.3) |
197 |
(192.7) |
570 |
(473.3) |
362 |
(369.0) |
128 |
(125.0) |
|
|
378 |
|
175 |
|
439 |
|
419 |
|
124 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 1. Cell viability analysis
Group |
Test article |
Average colony numbersa |
Relative survival (%)b |
With S9 Mix |
Negative controlc |
176.6 ± 2.7 |
100.0 |
Positive controld |
166.6 ± 6.7 |
94.3 ± 3.8 |
|
Test groups (mg/mL) |
|
|
|
2.0 |
198.0 ± 8.8 |
112.1 ± 5.0 |
|
1.0 |
218.0 ± 6.5 |
123.4 ± 3.7 |
|
0.5 |
199.2 ± 3.0 |
112.8 ± 1.7 |
|
0.25 |
203.8 ± 4.1 |
115.4 ± 2.3 |
|
Without S9 Mix |
Negative controlc |
196.0 ± 5.3 |
100.0 |
Positive controld |
140.2 ± 6.5 |
71.5 ± 3.3 |
|
Test groups (mg/mL) |
|
|
|
2.0 |
187.8 ± 6.4 |
95.8 ± 3.3 |
|
1.0 |
204.4 ± 4.0 |
104.3 ± 2.1 |
|
0.5 |
183.6 ± 2.7 |
93.7 ± 1.4 |
|
0.25 |
185.2 ± 1.6 |
94.5 ± 0.8 |
a Values were expressed as Mean ± S.D., and tests were repeated 5 times.
b Relative survival = each colony numbers of the positive control or test groups / the average of colony numbers in the negative control × 100%, then calculated the Mean ± S.D..
c Negative control: Ham’s F-12 medium with 1% DMSO and 10% FBS (S9 Mix or not).
d Positive control: 4 μg/mL B[a]P for the cell treated with S9 Mix, and 0.25 μg/mL 4-NQO for the cells treated without S9 Mix.
Table 2. Mutation frequency analysis
Group |
Test article |
Average colony numbersa |
Mutation frequency (× 10-6)b |
With S9 Mix |
Negative controlc |
2.8 ± 2.3 |
5.0 |
Positive controld |
91.6 ± 16.5 |
247.6* |
|
Test groups (mg/mL) |
|
|
|
2.0 |
3.2 ± 1.8 |
4.7 |
|
1.0 |
4.0 ± 2.0 |
8.9 |
|
0.5 |
3.2 ± 1.8 |
6.4 |
|
0.25 |
4.4 ± 3.0 |
6.1 |
|
Without S9 Mix |
Negative controlc |
13.0 ± 1.4 |
29.0 |
Positive controld |
42.6 ± 16.9 |
116.1* |
|
Test groups (mg/mL) |
|
|
|
2.0 |
19.6 ± 4.5 |
43.9 |
|
1.0 |
16.6 ± 4.2 |
33.5 |
|
0.5 |
10.2 ± 1.8 |
14.5 |
|
0.25 |
10.0 ± 2.0 |
17.5 |
a Values were expressed as Mean ± S.D., and tests were repeated 5 times.
b Mutation frequency = (average colony numbers of each test groups / the number of seeding) × (1 / Colonies formation frequency).
c Negative control: Ham’s F-12 medium with 1% DMSO and 10% FBS (S9 Mix or not).
dPositive control: 4μg/mL B[a]P for the cell treated with S9 Mix, and 0.25μg/mL 4-NQO for the cells treated without S9 Mix.
* Significantly different from the negative control group (ρ < 0.005).
Judgement |
Negative |
|
Reason for judgement and referential Matters |
No significant increase in the frequency of revertant colonies was recorded for any bacterial strain used in two separate experiments either with or without metabolic activation. An appropriate allowance of the purity of the test substance (93.7%) was made when the test material solution were prepared. A statistical analysis of the data was not required for the evaluation of the result. |
|
Test condition for the Plate method |
||
Composition |
Bacterial suspension 0.1 mL |
|
Test substance solution 0.1 mL |
||
Na-phosphate Buffer 0.5 mL |
||
S9 Mix (incase of metabolic activation method) 0.5 mL |
||
Top Agar solution: 2.0 mL |
||
Incubation |
Temperature/Time 37 °C/ 48 hours |
|
KEY TO TABLE OF RESULTS:
1. When bacterial growth is found, the applicable value is marked with an asterisk.
2. The average number of colonies for each concentration is recorded in parenthesis
3. “Number of revertants”: The observed values and average value are shown in order, beginning with the low concentration of the test substance.
4. The following postfixes are used when required:
C: Contaminated
P: precipitate
X: plate unscorable
ENNG= N-ethyl-N-nitro-N-nitrosoguanidine
4NQO= 4-nitroquinoline-N-oxide
9AA= 9-aminoacridine
2AA= 2-Aminoanthracene
BP= Benzo(a) pyrene
TABLE OF TEST RESULTS: EXPERIMENT I NAME OF THE TEST SUBSTANCE: KAYARUS SUPRA RUBINE BLN
|
|||||||||||
Number of revertants (number of colonies/plate) |
|||||||||||
With (+) S9 or Without (-) S9 |
Test substance concentration (ug/plate) |
Base-pair substitution type |
Fragmentg-type |
||||||||
0 |
TA 100 |
TA 1535 |
W2uvrA- |
TA 98 |
TA 1537 |
||||||
- |
72 |
|
15 |
|
13 |
16 |
|
15 |
|
|
|
100 |
(84.3) |
9 |
(11.7) |
19 (15.7) |
17 |
(16.7) |
16 |
(12.7) |
|
||
81 |
|
11 |
|
15 |
17 |
|
9 |
|
|
||
- |
8.0 |
61 |
|
13 |
|
14 |
16 |
|
14 |
|
|
87 |
(83.0) |
16 |
(14.7) |
13(13.7) |
14 |
(15.7) |
9 |
(10.0) |
|
||
101 |
|
15 |
|
14 |
17 |
|
7 |
|
|
||
- |
40 |
84 |
|
5 |
|
15 |
17 |
|
14 |
|
|
79 |
(76.7) |
13 |
(10.3) |
16(15.0) |
15 |
(16.0) |
10 |
(11.3) |
|
||
67 |
|
13 |
|
14 |
16 |
15 |
10 |
|
|
||
- |
200 |
89 |
|
13 |
|
11 |
15 |
|
14 |
|
|
88 |
(85.3) |
15 |
(14.3) |
13(11.3) |
16 |
16(15.7) |
9 |
(12.7) |
|
||
79 |
|
15 |
|
10 |
16 |
|
15 |
|
|
||
- |
1000 |
96 |
|
11 |
|
7 |
18 |
|
11 |
|
|
85 |
(86.9) |
10 |
(10.3) |
14(11.3) |
17 |
(17.3) |
6 |
(8.7) |
|
||
79 |
|
10 |
|
13 |
17 |
|
9 |
|
|
||
- |
5000 |
60 |
|
11 |
|
11 |
15 |
|
12 |
|
|
76 |
(74.3) |
11 |
(10.0) |
13(11.0) |
18 |
(16.0) |
16 |
(13.7) |
|
||
78 |
|
8 |
|
9 |
15 |
|
13 |
|
|
||
|
Name |
ENNG
|
ENNG |
ENNG |
4NQO |
9AA |
|||||
Positive control not requiring S9 mix |
Concentration (ug/plate) |
3.0
|
5.0 |
2.0 |
0.2 |
80.0 |
|||||
- |
Number of colonies/plate |
510 |
|
178 |
|
544 |
|
166 |
|
148 |
|
546 |
(534.7) |
189 |
(193.3) |
520 |
(534.0) |
156(158.3) |
|
152 |
(151.7) |
||
548 |
|
215 |
|
538 |
|
153 |
|
155 |
|
||
With (+) S9 or Without (-) S9 |
Test substance concentration (ug/plate) |
Base-pair substitution type |
Fragmentg-type |
|
|||||||
|
TA 100 |
TA 1535 |
W2uvrA- |
TA 98 |
TA 1537 |
|
|||||
+ |
0 |
131 |
|
14 |
|
19 |
|
17 |
|
12 |
|
123 |
(116.0) |
19 |
(15.7) |
19 |
(20.3) |
18 (17.7) |
|
15 |
(13.7) |
||
94 |
|
14 |
|
23 |
|
18 |
|
14 |
|
||
+ |
8.0 |
98 |
|
16 |
|
15 |
|
19 |
|
14 |
|
94 |
(100.7) |
11 |
(14.0) |
14 |
(14.0) |
22 (19.7) |
|
12 |
(14.0) |
||
110 |
|
15 |
|
13 |
|
18 |
|
16 |
|
||
+ |
40 |
106 |
|
11 |
|
12 |
|
19 |
|
16 |
|
99 |
(95.3) |
12 |
(10.3) |
9 |
(10.3) |
16 (16.3) |
|
14 |
(15.0) |
||
81 |
|
8 |
|
10 |
|
14 |
|
15 |
|
||
+ |
200 |
71 |
|
13 |
|
18 |
|
16 |
|
13 |
|
110 |
(85.3) |
7 |
(10.3) |
15 |
(17.3) |
15(17.3) |
|
10 |
(13.0) |
||
75 |
|
11 |
|
19 |
|
21 |
|
16 |
|
||
+ |
1000
|
73 |
|
16 |
|
7 |
|
12 |
|
10 |
|
108 |
(97.7) |
17 |
|
14 |
|
17 (14.7) |
|
11 |
(11) |
||
112 |
|
13 |
(15.3) |
15 |
(12.0) |
15 |
|
12 |
|
||
+ |
5000 |
85 |
|
8 |
|
13 |
|
14 |
|
9 |
|
71 |
(75.3) |
8 |
(7.3) |
21 |
(15.7) |
14(14.3) |
|
9 |
(8.7) |
||
70 |
|
7 |
|
13 |
|
15 |
|
8 |
|
||
|
Name |
BP
|
2AA
|
2AA |
BP
|
BP |
|||||
Positive control not requiring S9 mix |
Concentration (ug/plate) |
5.0
|
|
2.0 |
|
20.0 |
|
5.0 |
|
5.0 |
|
|
Number of colonies/plate |
339 |
|
206 |
|
505 |
|
326 |
|
123 |
|
|
|
343 |
(353.3) |
197 |
(192.7) |
570 |
(473.3) |
362 |
(369.0) |
128 |
(125.0) |
|
|
378 |
|
175 |
|
439 |
|
419 |
|
124 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 1. Cell viability analysis
Group |
Test article |
Average colony numbersa |
Relative survival (%)b |
With S9 Mix |
Negative controlc |
176.6 ± 2.7 |
100.0 |
Positive controld |
166.6 ± 6.7 |
94.3 ± 3.8 |
|
Test groups (mg/mL) |
|
|
|
2.0 |
198.0 ± 8.8 |
112.1 ± 5.0 |
|
1.0 |
218.0 ± 6.5 |
123.4 ± 3.7 |
|
0.5 |
199.2 ± 3.0 |
112.8 ± 1.7 |
|
0.25 |
203.8 ± 4.1 |
115.4 ± 2.3 |
|
Without S9 Mix |
Negative controlc |
196.0 ± 5.3 |
100.0 |
Positive controld |
140.2 ± 6.5 |
71.5 ± 3.3 |
|
Test groups (mg/mL) |
|
|
|
2.0 |
187.8 ± 6.4 |
95.8 ± 3.3 |
|
1.0 |
204.4 ± 4.0 |
104.3 ± 2.1 |
|
0.5 |
183.6 ± 2.7 |
93.7 ± 1.4 |
|
0.25 |
185.2 ± 1.6 |
94.5 ± 0.8 |
a Values were expressed as Mean ± S.D., and tests were repeated 5 times.
b Relative survival = each colony numbers of the positive control or test groups / the average of colony numbers in the negative control × 100%, then calculated the Mean ± S.D..
c Negative control: Ham’s F-12 medium with 1% DMSO and 10% FBS (S9 Mix or not).
d Positive control: 4 μg/mL B[a]P for the cell treated with S9 Mix, and 0.25 μg/mL 4-NQO for the cells treated without S9 Mix.
Table 2. Mutation frequency analysis
Group |
Test article |
Average colony numbersa |
Mutation frequency (× 10-6)b |
With S9 Mix |
Negative controlc |
2.8 ± 2.3 |
5.0 |
Positive controld |
91.6 ± 16.5 |
247.6* |
|
Test groups (mg/mL) |
|
|
|
2.0 |
3.2 ± 1.8 |
4.7 |
|
1.0 |
4.0 ± 2.0 |
8.9 |
|
0.5 |
3.2 ± 1.8 |
6.4 |
|
0.25 |
4.4 ± 3.0 |
6.1 |
|
Without S9 Mix |
Negative controlc |
13.0 ± 1.4 |
29.0 |
Positive controld |
42.6 ± 16.9 |
116.1* |
|
Test groups (mg/mL) |
|
|
|
2.0 |
19.6 ± 4.5 |
43.9 |
|
1.0 |
16.6 ± 4.2 |
33.5 |
|
0.5 |
10.2 ± 1.8 |
14.5 |
|
0.25 |
10.0 ± 2.0 |
17.5 |
a Values were expressed as Mean ± S.D., and tests were repeated 5 times.
b Mutation frequency = (average colony numbers of each test groups / the number of seeding) × (1 / Colonies formation frequency).
c Negative control: Ham’s F-12 medium with 1% DMSO and 10% FBS (S9 Mix or not).
dPositive control: 4μg/mL B[a]P for the cell treated with S9 Mix, and 0.25μg/mL 4-NQO for the cells treated without S9 Mix.
* Significantly different from the negative control group (ρ < 0.005).
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
No genotoxic effect was seen in the in vivo mouse micronucleeus test
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 1991-04-05 to 1991-06-11
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- read-across source
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Remarks:
- No significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups.
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE FINDING TOXICITY STUDY:
A range-finding study was performed to determine a suitable dose level for the micronucleus study. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of cytotoxicity up to a maximum recommended dose of 5000 mg/kg.
Groups of mice were dosed as follows:
DOSE LEVEL CONCENTRATION DOSE VOLUME NUMBER OF MICE
mg/kg mg/mL mL/kg Male Female
5000 500 10 7 7
All animals were dosed at once only by the appropriate dose level by gavage. The volume administered to each animal was calculated according to its fasted bodyweight at the time of dosing.
Animals were observed 1 hour after dosing and subsequently once daily for 3 days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.
The mortality data are summarised as follows:
DOSE LEVEL SEX NUMBER OF ANIMALS TREATED DEATH ON DAY TOTAL DEATH
0 1 2 3 1/14
5000 mg/kg Male 7 0 0 0 0
Female 7 1 0 0 0
No clinical signs were observed in any of the animals dosed with KAYARUS SUPRA RUBINE BLN.
There was only a premature death after one hour.
5000 mg/kg was selected as the maximum recommended dose for use in the micronucleus study.
RESULTS OF DEFINITIVE STUDY: see below.
- Statistical evaluation:If necessary, and where possible, all data were statistically analysed using appropriate statistical methods as recommended by the UKENS sub-committee and guidelines for mutagenicity testing Report part III (1989)
- Ratio of PCE/NCE (for Micronucleus assay):
There was no significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups. - Conclusions:
- Interpretation of results (migrated information): negative
The test material, Kayarus Supra Rubine BLN, was considered to be non-genotoxic under the conditions of the test. - Executive summary:
In order to assess the potential of the test substance to produce damage to chromosomes or anopleudias when administered oral to mice, a study was performed according to the OECD guidelines No 474 "Genetic toxicity: Micronucleus Test" and method B12 of Commission Directive 84/449/EEC.
Following a preliminary range finding study to confirm the oral toxicity of the test substance, the study was conducted using the test item at the maximum recommended dose level (5000 mg/kg)
In the micronucleus study a group of ten animals (5 male and 5 female) were given a single dose of the test substance. Animals were killed 24, 48 or 72 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.
Further groups of mice were dosed with arachis oil B.P. or cyclophosphamide, to serve as vehicle and positive controls, respectively.
Results:
Mortality Data and Clinical observations:
There were no premature deaths in any of the dose groups.
Clinical signs in animals dosed with the test substance: diarrhoea and coat stained with test substance.
Evaluation of Bone Marrow Slides:
There was no significant increase in the frequency of micronucleated PCE's in any of the test material dose groups when compared to their concurrent vehicle control groups, the increases
seen in the 72 and 24-hour sampling time groups were within accepted ranges.There was no significant increase in the frequency of micronucleated NCE's in any of the test material dose groups when compared to their concurrent vehicle control groups.
There was no significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups.
Based on the above findings and under the test conditions the test substance, was considered to be non-genotoxic under the conditions of the test.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1991-04-05 to 1991-06-11
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- endogenous gene animal assay
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Mice albino CD1 strain
to assess the potential of Kayarus Supra Rubine
BIN to produce damage to chromosomes or aneuploidy when administered
orally to mice. Groups of 10 mice
5 males (23-28g)
and 5 females (20-25g)
Approx. 5-8 weeks old
Minimum of acclimatisation period of 5 days
Gavage of a single oral dose of Kayarus Supra Rubine BLN at
5000 mg/kg.
Animals were killed 24, 48 or 72 hours later, the bone marrows extracted and smear preparations made and stained. Polychromatic
and normochromatic erythrocytes were scored for the presence of micronuclei.
Further groups of mice were dosed with arachis oil B.P. or cyclophosphamide, to serve as vehicle and positive controls respectively
- Route of administration:
- oral: gavage
- Vehicle:
- Oil Arachis B.P.
Identification and stability of the vehicle control were not determined. - Details on exposure:
- - Storage temperature of food: Room temperature
- Duration of treatment / exposure:
- Animals were observed 1 hour after dosing and subsequently once daily for 3 days. Any deaths and evidence of overt toxicity were recorded at each observation.
- Frequency of treatment:
- Once daily for 3 days
- Dose / conc.:
- 5 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Range finding Toxicity Study:
Dose level: 5000 mg/kg
Concentration: 500 mg/mL
Dose Volume: 10 ml/kg
No. of male 5
No. of female 5 - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide Monohydrate was freshly prepared as required as a solution at the appropriate concentration in distilled water. Identification and stability of control material and the preparations were not determined.
- Tissues and cell types examined:
- Erythrocytes (micronucleaded polychromatic and normochromatic)
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: Range finding study
DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice (i.e. 24, 48 or 72 hours), one femur was dissected and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixe in absolute methanol, and stained in May-Grünwald/Giemsa.
EVALUATION OF SLIDES
Stained bone marrow smears were examined at random using light microscopy at x 1000 magnification. The incidence of micronucleated cells per 1000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored.
Micronuclei are normally circular in shape, although occasionally they may be oval or half moon shaped, and have a sharp contour with even staining. In additional the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 polychromatic erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of normochromatic to polychromatic erythrocytes was calculated together with appropriate group mean values for males and females separately and combined. - Evaluation criteria:
- There are several criteria for determining a positive result, such as:
- a dose-related increase in the number of micronucleated cells or
-a clear increase in the number of micronucleated cells in a single dose group at a single sampling time.
A test substance for which the results do not meet the above criteria is considered nonmutagenic in this test.
-Biological relevance of the results.
-Statistical significance
A positive response for bone marrow toxicity is demonstrated when the dose group mean normochromatic to polychromatic ratio is shown to be statistically significant from the concurrent vehicle control group. - Statistics:
- If necessary, and where possible, all data were statistically analysed using appropriate statistical methods as recommended by the UKENS sub-committee and guidelines for mutagenicity testing Report part III (1989)
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Remarks:
- No significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups.
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE FINDING TOXICITY STUDY:
A range-finding study was performed to determine a suitable dose level for the micronucleus study. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of cytotoxicity up to a maximum recommended dose of 5000 mg/kg.
Groups of mice were dosed as follows:
DOSE LEVEL CONCENTRATION DOSE VOLUME NUMBER OF MICE
mg/kg mg/mL mL/kg Male Female
5000 500 10 7 7
All animals were dosed at once only by the appropriate dose level by gavage. The volume administered to each animal was calculated according to its fasted bodyweight at the time of dosing.
Animals were observed 1 hour after dosing and subsequently once daily for 3 days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.
The mortality data are summarised as follows:
DOSE LEVEL SEX NUMBER OF ANIMALS TREATED DEATH ON DAY TOTAL DEATH
0 1 2 3 1/14
5000 mg/kg Male 7 0 0 0 0
Female 7 1 0 0 0
No clinical signs were observed in any of the animals dosed with KAYARUS SUPRA RUBINE BLN.
There was only a premature death after one hour.
5000 mg/kg was selected as the maximum recommended dose for use in the micronucleus study.
RESULTS OF DEFINITIVE STUDY: see below.
- Statistical evaluation:If necessary, and where possible, all data were statistically analysed using appropriate statistical methods as recommended by the UKENS sub-committee and guidelines for mutagenicity testing Report part III (1989)
- Ratio of PCE/NCE (for Micronucleus assay):
There was no significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups. - Conclusions:
- Interpretation of results (migrated information): negative
The test material, Kayarus Supra Rubine BLN, was considered to be non-genotoxic under the conditions of the test. - Executive summary:
In order to assess the potential of the test substance to produce damage to chromosomes or anopleudias when administered oral to mice, a study was performed according to the OECD guidelines No 474 "Genetic toxicity: Micronucleus Test" and method B12 of Commission Directive 84/449/EEC.
Following a preliminary range finding study to confirm the oral toxicity of Kayarus Supra Rubine BLN, the study was conducted using the test item at the maximum recommended dose level (5000 mg/kg)
In the micronucleus study a group of ten animals (5 male and 5 female) were given a single dose of the test substance.Animals were killed 24, 48 or 72 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.
Further groups of mice were dosed with arachis oil B.P. or cyclophosphamide, to serve as vehicle and positive controls, respectively.
Results:
Mortality Data and Clinical observations:
There were no premature deaths in any of the dose groups.
Clinical signs in animals dosed with Kayarus Supra Rubine BLN: diarrhoea and coat stained with test substance.
Evaluation of Bone Marrow Slides:
There was no significant increase in the frequency of micronucleated PCE's in any of the test material dose groups when compared to their concurrent vehicle control groups, the increases
seen in the 72 and 24-hour sampling time groups were within accepted ranges.There was no significant increase in the frequency of micronucleated NCE's in any of the test material dose groups when compared to their concurrent vehicle control groups.
There was no significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups.
Based on the above findings and under the test conditions the test substance, was considered to be non-genotoxic under the conditions of the test.
Referenceopen allclose all
Test design:
Dose Group |
Dose Level mg/kg |
Concentration mg/mL |
Dose Volume ml/kg |
Kill time (hours after dosing) |
Animal
Male |
Numbers
female |
Vehicle control (Arachis oil B.P.) |
0 |
0 |
10 |
72 |
1-5 |
6-10 |
Vehicle control (Arachis oil B.P.) |
0 |
0 |
10 |
48 |
11-15 |
16-20 |
Vehicle control (Arachis oil B.P.) |
0 |
0 |
10 |
24 |
21-25 |
26-30 |
Positive Control (Cyclophosphamide) |
50 |
5 |
10 |
24 |
31-35 |
36-40 |
Kayarus Supra Rubine BLN |
5000 |
500 |
10 |
72 |
41-45 |
46-50 |
Kayarus Supra Rubine BLN |
5000 |
500 |
10 |
48 |
51-55 |
56-60 |
Kayarus Supra Rubine BLN |
5000 |
500 |
10 |
24 |
61-65 |
66-70 |
Comparison was made between no. of micronucleated polychromatic erythrocytes occurring in each of the three test material groups and the no occurring in the corresponding vehicle control groups.
A positive mutagenic response is demonstrated when a statistically significant increasing the number of micronucleated polychromatic erythrocytes is observed for ether of the kill times.
A positive response of bone marrow toxicity is demonstrated when the dose group mean normochromatic to polychromatic ratio is shown to be statistically significant from the concurrent vehicle control group.
Statistical methods, if necessary: which are recommended by the UKEMS sub-committee on guidelines for mutagenicity testing, Report, part III (1989)
Results:
Mortality Data and Clinical observations:
There were no premature deaths in any of the dose groups.
Clinical signs in animals dosed with Kayarus Supra Rubine BLN: diarrhoea and coat stained with test substance.
Evaluation of Bone Marrow Slides:
There was
no significant increase in the frequency of micronucleated PCE's in
any of the test material dose groups when compared to their concurrent
vehicle control groups, the increases
seen in the 72 and 24-hour sampling time groups were within accepted
ranges.
There was no significant increase in the frequency of micronucleated NCE's in any of the test material dose groups when compared to their concurrent vehicle control groups.
There was no significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups.
The
positive control group showed a marked increase in the incidence of
micronucleated polychromatic erythrocytes hence
confirming the sensitivity of the system to the known mutagenic activity
of cyclophosphamide under the conditions of the test. The test material,
Kayarus Supra Rubine BLN, was found not to produce micronuclei in
polychromatic erythrocytes of mice under the conditions of the test.
Test design:
Dose Group |
Dose Level mg/kg |
Concentration mg/mL |
Dose Volume ml/kg |
Kill time (hours after dosing) |
Animal
Male |
Numbers
female |
Vehicle control (Arachis oil B.P.) |
0 |
0 |
10 |
72 |
1-5 |
6-10 |
Vehicle control (Arachis oil B.P.) |
0 |
0 |
10 |
48 |
11-15 |
16-20 |
Vehicle control (Arachis oil B.P.) |
0 |
0 |
10 |
24 |
21-25 |
26-30 |
Positive Control (Cyclophosphamide) |
50 |
5 |
10 |
24 |
31-35 |
36-40 |
Kayarus Supra Rubine BLN |
5000 |
500 |
10 |
72 |
41-45 |
46-50 |
Kayarus Supra Rubine BLN |
5000 |
500 |
10 |
48 |
51-55 |
56-60 |
Kayarus Supra Rubine BLN |
5000 |
500 |
10 |
24 |
61-65 |
66-70 |
Comparison was made between no. of micronucleated polychromatic erythrocytes occurring in each of the three test material groups and the no occurring in the corresponding vehicle control groups.
A positive mutagenic response is demonstrated when a statistically significant increasing the number of micronucleated polychromatic erythrocytes is observed for ether of the kill times.
A positive response of bone marrow toxicity is demonstrated when the dose group mean normochromatic to polychromatic ratio is shown to be statistically significant from the concurrent vehicle control group.
Statistical methods, if necessary: which are recommended by the UKEMS sub-committee on guidelines for mutagenicity testing, Report, part III (1989)
Results:
Mortality Data and Clinical observations:
There were no premature deaths in any of the dose groups.
Clinical signs in animals dosed with Kayarus Supra Rubine BLN: diarrhoea and coat stained with test substance.
Evaluation of Bone Marrow Slides:
There was
no significant increase in the frequency of micronucleated PCE's in
any of the test material dose groups when compared to their concurrent
vehicle control groups, the increases
seen in the 72 and 24-hour sampling time groups were within accepted
ranges.
There was no significant increase in the frequency of micronucleated NCE's in any of the test material dose groups when compared to their concurrent vehicle control groups.
There was no significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups.
The
positive control group showed a marked increase in the incidence of
micronucleated polychromatic erythrocytes hence
confirming the sensitivity of the system to the known mutagenic activity
of cyclophosphamide under the conditions of the test. The test material,
Kayarus Supra Rubine BLN, was found not to produce micronuclei in
polychromatic erythrocytes of mice under the conditions of the test.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
A genetic toxicity test was performed to assess the mutagenic potential of the test substance using a bacterial/microsome test system. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA 100, TA100, TA 1535 and TA 1537) and the trytophan-requiring auxotroph strains of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from rat liver.
Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems, not available in the bacterial cell, the test material and the bacteria were also incubated in the presence of a liver microsomal fraction (S9) obtained from rats previously treated with a compound known to induce and elevated level of these enzyme.
The test study included a:
1) Preliminary toxicity study
The dose range used in the preliminary toxicity study was 0, 312.5, 625, 1250, 2500 and 5000 µg/plate. The test material was non-toxic in the strains of bacteria used (TA100 and WP2uvrA).
2) Mutation study
The results for the checks on characteristics, viability spontaneous reversion rate for each tester strain were all found to be satisfactory.
No toxicity was exhibited to any of the strains of bacteria used.
No significant increase in the frequency of revertant colonies was recorded for any bacterial strain used in two separate experiments either with or without metabolic activation. An appropriate allowance of the purity of the test substance was made when the test material solution were prepared. A statistical analysis of the data was not required for the evaluation of the result.
The positive control substance all produced marked increased in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.
Therefore the test substancewas found to be non-mutagenic under the conditions of this test
The in vitro mammalian cell gene mutation test was performed in accordance with OECD 476: 2015 in order to evaluate the test substance for its cytotoxicity and mutation frequency. This test used Chinese hamster ovary cells (CHO-K1). The cytotoxicity determination of the test article for CHO-K1 was used to set the maximum dosage and number of seeding cells in the gene mutation test. In the gene mutation test, test substance concentrations of 250, 500, 1000, and 2000 µg/ml were incubated for 3 hours in the presence or absence of rat liver S9 Mix. The test article maximum concentration of 2.0 mg/mL was determined in a cell viability test where all survival rates for this concentration were greater than 50%. By statistical analysis, the mutation frequency in each concentration of the test article for 3 hours treatment with or without S9 Mix was not significantly different from the negative control group. Therefore, under these test conditions of this study, the test substance was negative in the in vitro mammalian cell gene mutation test.
In order to assess the potential of the test substance to produce damage to chromosomes or anopleudias when administered oral to mice, a study was performed according to the OECD guidelines No 474 "Genetic toxicity: Micronucleus Test" and method B12 of Commission Directive 84/449/EEC.
Following a preliminary range finding study to confirm the oral toxicity of the test substance, the study was conducted using the test item at the maximum recommended dose level (5000 mg/kg)
In the micronucleus study a group of ten animals (5 male and 5 female) were given a single dose of the test substance.Animals were killed 24, 48 or 72 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.
Further groups of mice were dosed with arachis oil B.P. or cyclophosphamide, to serve as vehicle and positive controls, respectively.
There were no premature deaths in any of the dose groups. Clinical signs in animals dosed with the test substance comprised of diarrhoea and coat stained with test substance.
There
was no significant increase in the frequency of micronucleated PCE's in
any of the test material dose groups when compared to their concurrent
vehicle control groups, the increases
seen in the 72 and 24-hour sampling time groups were within accepted
ranges.
There was no significant increase in the frequency of micronucleated NCE's in any of the test material dose groups when compared to their concurrent vehicle control groups.
There was no significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups.
Based on the above findings and under the test conditions the test substance, was considered to be non-genotoxic under the conditions of the test.
Justification for classification or non-classification
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