Registration Dossier
Registration Dossier
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EC number: 221-424-4 | CAS number: 3089-17-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Pigment red 202 (not specified form): negative (Ames)
Pigment red 202 (not specified form): negative (MLA)
Structural analogue:
Pigment violet 19 (nano form): negative (Ames)
Pigment violet 19 (nano form): negative (HGPRT)
Pigment violet 19 (nano form): negative (Chromosomal Aberration)
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
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).
4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13). - Reason / purpose for cross-reference:
- read-across source
- Vehicle / solvent:
- - Vehicle used: DMSO
- Species / strain:
- other: S. typhimurium TA 1535, TA 98, TA 100 and TA 1537, E. coli WP2uvrA
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- minor effect in plate incorporation assay at 5000 µg/plate in TA1537 (reduction in the number of revertants to ~50% of control plates)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: visible in Experiment II without S9 at a concentration of 5000 µg/plate and with S9-Mix at concentrations of 1000 µg/plate and above
COMPARISON WITH HISTORICAL CONTROL DATA:
- historical range of positive controls was exceeded with metabolic activation in strains TA 98 and TA 1537 (Experiment I) and in strain TA 100 (Experiment/ and II), this effect indicates the sensitivity of the strains rather than compromising the assay
- Strain WP2uvrA (ExperimentI) with metabolic activation historical control range of negative and solvent controls was slightly exceeded
- Strain WP2uvrA and TA 1535 (ExperimentII) with and without metabolic activation historical control range of negative and solvent controls was slightly exceeded
- Strain TA 1535 and TA 98 (ExperimentII) with metabolic activation historical control range of negative and solvent controls was slightly exceeded
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- TA 1537: plate incorporation assay without metabolic activation minor effect at 5000 µg/plate - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- The toxicity potential of Pigment Red 202 is assessed based on analogue approaches.
Interpretation of results:
negative with metabolic activation
negative without metabolic activation
The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and preincubation assay) with and without metabolic activation. - Executive summary:
Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA100 and Escherichia coli strain WP2uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate using the plate incorporation assay. Additionally, a preincubation assay with or without metabolic activation was performed using the concentrations 33, 100, 333, 1000, 2500, and 5000 µg/plate.
The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.
- 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
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).
4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13). - Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- other: S. Typhimurium TA 98 and E.coli WP2uvrA
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- minor effect in Experiment I without metabolic activation at a concentration of 2500 µg/plate and above (reduction in the number of revertants to ~50% of control plates)
- 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:
- cytotoxicity
- Remarks:
- minor effect in Experiment I without metabolic activation at a concentration of 5000 µg/plate and with metabolic activation at a concentration of 2500 µg/plate and above (reduction in the number of revertants to ~50% of control plates)
- 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:
- cytotoxicity
- Remarks:
- minor effect in Experiment I and II each with metabolic activation at a concentration of 5000 µg/plate (reduction in the number of revertants to ~50% of control plates)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The toxicity potential of Pigment Red 202 is assessed based on analogue approaches.
Interpretation of results:
negative with metabolic activation
negative without metabolic activation
The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and preincubation assay) with and without metabolic activation.
The findings of this study support the results given in the key study. - Executive summary:
Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA100 and Escherichia coli strain WP2uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate using the plate incorporation assay. Additionally, a preincubation assay with or without metabolic activation was performed using the concentrations 33, 100, 333, 1000, 2500, and 5000 µg/plate.
The test item did not reveal any mutagenic activity under the conditions tested.The appropriate reference mutagenes showed distinct positive mutagenic effects.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).
4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13). - Reason / purpose for cross-reference:
- read-across source
- Statistics:
- no data
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations of >= 3500 µg/ml
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
A pretest was performed to determine toxic concentrations of the test item (9.77-5000 µg/ml). 5000 µg/ml were cytotoxic with or without metabolic activation, the lower concentrations were nontoxic.
COMPARISON WITH HISTORICAL CONTROL DATA:
The results of vehicle and positive controls were in the range of historical controls
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- The toxicity potential of Pigment Red 202 is assessed based on analogue approaches.
Interpretation of results:
negative with metabolic activation
negative without metabolic activation
The test item (2,9-dichloroquinacridone) did not exert mutagenic activity in the L5178Y TK+/TK- mouse lymphoma mutation assay with and without metabolic activation. - Executive summary:
The test item was examined for the ability to induce forward mutations at the thymidine kinase (TK) locus in the L5178Y mouse lymphoma cell line.The mutation assays were performed at concentrations up to 5000 µg/ml with and without metabolic activation (S9 liver homogenate from Aroclor-induced rats), based on the results of a cytotoxicity pretest.
Without metabolic activation, treatments at 3500 µg/ml and 5000 µg/ml were moderately cytotoxic and the remaining four treatments (78.1 µg/ml to 1250µg/ml) were noncytotoxic. None of the six analyzed treatments induced a mutant frequency that exceeded the minimum criterion for a positive response. In the presence of metabolic activation, treatments from 78.1 µg/ml to 5000 µg/ml were also evaluated. Concentrations of 3500 µg/ml and 5000 µg/ml were weakly cytotoxic and the remaining four treatments were noncytotoxic. No evidence for mutagenicity was observed at any concentration.
- 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
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).
4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13). - Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- - Precipitation: the test item formed a suspension in DMSO in the stock solution and all dilutions tested
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- The toxicity potential of Pigment Red 202 is assessed based on analogue approaches. Interpretation of results:
negative with metabolic activation
negative without metabolic activation
The test item (2,9-dichloroquinacridone) did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay) with and without metabolic activation. - Executive summary:
Mutagenic activity of the test item (2,9-dichloroquinacridone) was investigated in Escherichia coliWP2uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate using the plate incorporation assay. The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.
- 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
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).
4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13). - Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- - Precipitation: the test item formed a suspension in DMSO in the stock solution and all dilutions tested and precipitated at the highest concentration tested (5000 µg/plate)
RANGE-FINDING/SCREENING STUDIES:
The dose rangefinding study was conducted on the test article using tester strain TA100 in both the presence and absence of S9 (one plate per dose). Ten doses of test article, from 5,000 to 6.67 µg were tested. No cytotoxicity was observed in either the presence or absence of S9 as evidenced by no observed decrease in the number of revertants per plate. The bacterial background lawn was evaluated as normal up to the 1,000 pg per plate dose in both the presence and absence of S9. The lawns on the plates above this dose could not be evaluated due to the presence of test article precipitate. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
The test item (91235) did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay) with and without metabolic activation. - Executive summary:
Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 with (induced rat liver S9 mix) and without metabolic activation at concentrations of 100, 333, 667, 1000, 3330 and 5000 µg/plate using the plate incorporation assay. The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.
- Endpoint:
- genetic toxicity in vitro, other
- Remarks:
- Mammalian Cell Gene Mutation Tests
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02 July 2021 to 04 November 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- JUSTIFICATION FOR DATA WAIVING
[Specific explanation in addition to field 'Justification for data waiving'] - Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted on 29th July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes
- Target gene:
- CHO AA8 cells
- Species / strain / cell type:
- other: CHO AA8 cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: American Type Culture Collection (ATCC)
- Cell doubling time : Approximately 12 hours
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Alpha MEM CULTURE MEDIA with 10% FBS and 1% penstrep and 5±1% CO2
- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Doubling time model chromosome number cells were checked. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 homogenate
- Test concentrations with justification for top dose:
- 15.625, 31.25, 62.5 and 125 µg/mL based on initial cytotoxicity test
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- benzo(a)pyrene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 2×107
DURATION
- Exposure duration: 3 hours and 38 minutes
- Expression time (cells in growth medium): 7 days for mutant phenotype expression
- Selection time (if incubation with a selection agent):8 days
SELECTION AGENT (mutation assays): 10 µM of 6-Thioguanine
STAIN : 5% giemsa
NUMBER OF REPLICATIONS: 5
METHODS STAINING TECHNIQUE USED: Post incubation period, medium from each culture flask was aspirated and stained with 5% Giemsa stain. Number of colonies formed was counted manually.
DETERMINATION OF CYTOTOXICITY
- Method:cloning efficiency - Rationale for test conditions:
- Acceptance of a test is based on the following criteria:
•The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database as described in OECD guidelines for testing of chemicals, No. 476.
•Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control.
•Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
•Adequate number of cells and concentrations are analysable (according to OECD guidelines for testing of chemicals, No. 476).
•The criteria for the selection of top concentration are consistent with those described in OECD guidelines for testing of chemicals, No. 476. - Evaluation criteria:
- Colony counts in selective media and non-selective media
- Statistics:
- yes, Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0.
- Key result
- Species / strain:
- other: CHO AA8 cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change in pH was observed in any of the test concentrations
- Water solubility: Insoluble
- Precipitation: The precipitation and pH were tested at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL concentrations. Post 3 hours of incubation, no precipitation was observed at the tested concentrations of 0.03125, 0.0625 mg/mL, moderate precipitation was observed at 0.125 and 0.25 mg/mL, heavy precipitation was observed at 0.5, 1 and 2 mg/mL
RANGE-FINDING/SCREENING STUDIES: Yes
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Positive Control- Benzo(a)pyrene and 4- Nitroquinoline N-oxide
With Metabolic Activation
(3 to 6 hours)
[Benzo(a)pyrene] Without Metabolic Activation
(3 to 6 hours)
[4 Nitroquinoline N-oxide]
Mean Data of Mutant Frequency/2x106 Cells 261.94 264.60
Standard
Deviation 27.28 18.52
Margin of Error 17.82 12.10
Upper bound 279.76 276.70
Lower bound 244.12 252.50
- Negative (solvent/vehicle) historical control data: Negative controls were maintained.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Relative survivality - Conclusions:
- The test item is considered as non-mutagenic at and up to the concentration of 125 µg/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 formed a suspension in acetone at concentration of 200 mg/mL. After 3 hours of incubation, no precipitation was observed at the tested concentrations of 0.03125 and 0.0625 mg/mL, moderate precipitation was observed at 0.125 and 0.25 mg/mL, and heavy precipitation was observed at 0.5, 1 and 2 mg/mL. No change in pH was observed in any of the test concentrations.
On the basis of precipitation results, 0.125 mg/mL (125 µg/mL) was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 7.8125, 15.625, 31.25, 62.5 and 125 µg/mL using acetone as a vehicle in four flasks/group in the presence and absence of metabolic activation (4 hours and 5 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% (62.61 % in presence of metabolic activation and64.60 % in absence of metabolic activation) at 125 µg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 125 µg/mL was selected as highest concentration for gene mutation test.
The gene mutation test was conducted at the concentrations of 125, 62.5, 31.25 and 15.625 µg/mL using acetone as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 38 minutes).Benzo(a) pyrene and4 Nitroquinoline N-oxidewere used aspositive controlsfor the gene mutation test.
Cytotoxicity as Relative Survival was 64.10 % in presence of metabolic activation and63.48 % in absence of metabolic activationat the highest tested concentration of 125µg/mL. Relative Survival of negative control was 100.00 in presence of metabolic activation and 103.48 in absence of metabolic activation.There was no statistically significant increase in mutant frequencies at any of the concentrations tested and negative control when compared with the vehicle control.
There was statistically significant increase in mutant frequenciesfor positive controlswhen compared with the vehicle controlin bothmetabolic activation andabsence 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:
- 20 January 2021 to 06 May 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- OECD 473
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted on 29th July 2016.
- Deviations:
- no
- Principles of method if other than guideline:
- - Principle of test:
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the solvent/vehicle: acetone - Target gene:
- Human Peripheral Blood Lymphocytes were used
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- Human Peripheral Blood Lymphocytes were used
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 homogenate
- Test concentrations with justification for top dose:
- 0.015625, 0.03125 and 0.0625 mg/mL, top dose selected based on initial cytotoxicity results
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: formed suspension in 200 mg/mL - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium - RPMI
DURATION
- Exposure duration: 3 to 6 hours and 24 hours
SELECTION AGENT (mutation assays): RPMI 1640 MEDIUM
STAIN : Giemsa
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Pellets were mixed with 4 to 5 mL of freshly prepared warm 0.56% Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 4 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cell suspension was incubated for 10 minutes at room temperature and later suspension was centrifuged at 2200 rpm for 10 minutes. The procedure was repeated twice by adding 4 mL of cold acetic acid: methanol fixative (1:3).
Clean slides were stored in a beaker with distilled water and kept in the refrigerator for at least 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension were aspirated and dropped onto the chilled slide pre labeled with study number, with (+S9) or without metabolic activation (-S9), treatment/group and slide number. The slides were air dried
NUMBER OF CELLS EVALUATED: 150 metaphase
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - Rationale for test conditions:
- • Three analyzable concentrations were used for chromosomal aberration test.
• Initial cytotoxicity was conducted at concentrations of 0.015625, 0.03125, 0.0625 and 0.125 mg/mL. The percentage reduction in Mitotic Index was in the range of 10.00 to 45.61 at 0.0625 mg/mL. As the percentage reduction in MI was not more than 45±5% at 0.0625 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.015625 and 0.03125 mg/mL - Evaluation criteria:
- Percentage mitotic index
- Statistics:
- yes
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change in pH was observed in any of the concentrations tested upto 2 mg/mL
- Water solubility: insoluble
- Precipitation: Precipitation test was conducted at 0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 24 hours of incubation, heavy precipitation was observed at 0.5, 1 and 2 mg/mL, moderate precipitation was observed at 0.125, 0.25 mg/mL and mild precipitation was observed at 0.0625 mg/mL. No precipitation was observed at 0.03125 and 0.015625 mg/mL
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: NA
- Negative (solvent/vehicle) historical control data: NEGATIVE CONTROLS MAINTAINED. - Conclusions:
- Based on the results obtained, the test item is considered as non-clastogenic upto the concentration of 0.0625 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 suspension in acetone at 200 mg/mL. Precipitation and pH test was conducted at 0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 24 hours of incubation, heavy precipitation was observed at 0.5, 1 and 2 mg/mL, moderate precipitation was observed at 0.25, 0.125 mg/mL, mild precipitation was observed at 0.625 mg/mL. No precipitation was observed at 0.03125 and 0.015625 mg/mL. No change in pH was observed in any of the concentrations tested upto 2 mg/mL. Hence, 0.125 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.015625, 0.03125 and 0.0625 mg/mL of test item.
In initial cytotoxicity test, the percentage reduction in Mitotic Index was in the range of 10.00 to 45.61 at 0.015625, 0.03125 and 0.0625mg/mL (as there were colored particles and precipitation was interfering with the cells, the slides could not be evaluated at 0.125 mg/mL) in presence of metabolic activation (short term), in absence of metabolic activation (short term) in absence of metabolic activation (long term), respectively. As the percentage reduction inmitotic index was not more than 45±5% at 0.0625 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. other concentrations tested were 0.015625 and 0.03125 mg/mL.
In the chromosomal aberration test, the cells were treated with test item at the concentrations of 0.015625, 0.03125 and 0.0625mg/mLusing acetone 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.
Theconcurrent 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 mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 28 FEB 1995 to 22 MAR 1995
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study (OECD 476) with some restrictions (insufficient number of single cultures examined, no colony sizing performed)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- : only 6 of 8 single cultures evaluated, no colony sizing performed
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- thymidine kinase locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
Growth medium: supplemented RPMI 1640
Treatment medium: supplemented Fishers medium, with 10% Pluronic F-68
Cloning medium: supplemented Fishers medium, with 0.24% purified agar, without Pluronic F-68
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes (cultivation with aminopterin or methotrexate) - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver homogenate from Aroclor 1254 induced Sprague-Dawley rats
- Test concentrations with justification for top dose:
- without metabolic activation: 78.1, 156, 313, 1250, 3500, 5000 µg/ml
with metabolic activation: 78.1, 156, 625, 1250, 3500, 5000 µg/ml - Vehicle / solvent:
- 10% Pluronic F68
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- Migrated to IUCLID6: without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- Remarks:
- Migrated to IUCLID6: with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
Single experiments in triplicates were performed
DURATION
- Exposure duration: about 4 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 10-14 days
SELECTION AGENT (mutation assays): 5-trifluorothymidine (3 µg/ml)
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth - Evaluation criteria:
- A test article is evaluated as nonmutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing ten to twenty percent relative growth or, in the case of relatively nontoxic materials, a range of applied concentrations extending to the maximum of 5000 µg/ml.
The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that is 2 times the concurrent background mutant frequency. The background mutant frequency is defined as the average mutant frequency of the vehicle control cultures. A dose-related or toxicity-related increase in mutant frequency should be observed. It is desirable to obtain this relation for at least three doses, but this depends upon the concentration steps chosen for the assay and the toxicity at which mutagenic activity appears. If the mutant frequency obtained for a single dose at or near the highest testable toxicity is about two or more times the minimum criterion, the test material will be considered mutagenic in a single trial. Smaller increases at a single dose near the highest testable toxicity will require confirmation by a repeat assay. - Statistics:
- no data
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations of >= 3500 µg/ml
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
A pretest was performed to determine toxic concentrations of the test item (9.77-5000 µg/ml). 5000 µg/ml were cytotoxic with or without metabolic activation, the lower concentrations were nontoxic.
COMPARISON WITH HISTORICAL CONTROL DATA:
The results of vehicle and positive controls were in the range of historical controls
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
The test item (2,9-dichloroquinacridone) did not exert mutagenic activity in the L5178Y TK+/TK- mouse lymphoma mutation assay with and without metabolic activation. - Executive summary:
The test item was examined for the ability to induce forward mutations at the thymidine kinase (TK) locus in the L5178Y mouse lymphoma cell line.The mutation assays were performed at concentrations up to 5000 µg/ml with and without metabolic activation (S9 liver homogenate from Aroclor-induced rats), based on the results of a cytotoxicity pretest.
Without metabolic activation, treatments at 3500 µg/ml and 5000 µg/ml were moderately cytotoxic and the remaining four treatments (78.1 µg/ml to 1250µg/ml) were noncytotoxic. None of the six analyzed treatments induced a mutant frequency that exceeded the minimum criterion for a positive response. In the presence of metabolic activation, treatments from 78.1 µg/ml to 5000 µg/ml were also evaluated. Concentrations of 3500 µg/ml and 5000 µg/ml were weakly cytotoxic and the remaining four treatments were noncytotoxic. No evidence for mutagenicity was observed at any concentration.- 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:
- From 23 FEB 1995 to 4 MAR 1995
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study (OECD 471) with acceptable restrictions (not all proposed strains tested)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- : only E. coli strain WP2 uvrA tested
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver homogenate from Aroclor 1254 induced Sprague-Dawley rats
- Test concentrations with justification for top dose:
- 33.3, 66.7, 100, 333, 667, 1000, 3330, 5000 µg/plate
- Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: without metabolic activation: 4-nitroquinoline-oxide; with metabolic activation: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
The tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al (1975) and Maron and Ames (1983). All doses of the test article, the vehicle controls and the positive controls were plated in triplicate.
The S9 mix and dilutions of the test article were prepared immediately prior to their use.
When S9 mix was not required, 100 µl of tester strain and 100 µl of vehicle or test article dose was added to 2.5 ml of molten selective top agar (maintained at 45 ± 2°C). When S9 mix was required, 500 µl of S9 mix, 100 µl of tester strain and 100 ml of vehicle or test article dose was added to 2.0 ml of molten selective top agar. After the required components had been added, the mixture was vortexed and overlaid onto the surface of 25 ml of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay had solidified, the plates were inverted and incubated for 48 ± 8 hr at 37 ± 2°C. Positive control articles were plated using a 50 µl plating aliquot.
DURATION
- Exposure duration: 48 +/- 8 h
DETERMINATION OF CYTOTOXICITY
- Method: other: decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn. - Evaluation criteria:
- - Positive Control Values in the Absence of S9 Mix
To demonstrate that the tester strain were capable of identifying a mutagen, the mean value of a positive control for the tester strain exhibited at least a 3-fold increase over the mean value of the vehicle control for WP2uvrA.
- Positive Control Values in the Presence of S9 Mix (S9 Mix Integrity)
To demonstrate that the S9 mix was capable of metabolizing a promutagen to its mutagenic form(s), the mean value of the positive control for a respective tester strain in the presence of the S9 mix exhibited at least a 3-fold increase over the mean value of the vehicle control for WP2uvrA.
- Criteria For A Positive Response
For a test article to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of the tester strain over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article. - Statistics:
- no data
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- - Precipitation: the test item formed a suspension in DMSO in the stock solution and all dilutions tested
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
The test item (2,9-dichloroquinacridone) did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay) with and without metabolic activation. - Executive summary:
Mutagenic activity of the test item (2,9-dichloroquinacridone) was investigated in Escherichia coliWP2uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate using the plate incorporation assay. The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.
- 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:
- From 21 JUL 1992 to 4 SEP 1992
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study (OECD 471) with acceptable restrictions (not all proposed strains tested)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- : proposed strains E.coli WP2 uvrA or S.typhimurium TA102 not tested
- 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
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 1538
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver homogenate from Aroclor 1254 induced Sprague-Dawley rats
- Test concentrations with justification for top dose:
- 100, 333, 667, 1000, 3330, 5000 µg/plate
- Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: without metabolic activation: sodium azide (TA100, TA1535); 2-nitrofluorene (TA98, TA1538); ICR-191 (TA1537); with metabolic activation: 2-aminoanthracene (TA98, TA100, TA1535, TA1537, TA1538)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
A dose rangefinding study was performed using tester strain TA100 both in the presence and absence of S9 mix. Ten doses of test article were tested at one plate per dose. The test article was checked for cytotoxicity up to a maximum concentration of 5000 µg/plate.
The tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al (1975) and Maron and Ames (1983). All doses of the test article, the vehicle controls and the positive controls were plated in triplicate.
The S9 mix and dilutions of the test article were prepared immediately prior to their use.
When S9 mix was not required, 100 µl of tester strain and 100 µl of vehicle or test article dose was added to 2.5 ml of molten selective top agar (maintained at 45 ± 2°C). When S9 mix was required, 500 µl of S9 mix, 100 µl of tester strain and 100 ml of vehicle or test article dose was added to 2.0 ml of molten selective top agar. After the required components had been added, the mixture was vortexed and overlaid onto the surface of 25 ml of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay had solidified, the plates were inverted and incubated for 48 ± 8 hr at 37 ± 2°C. Positive control articles were plated using a 50 µl plating aliquot.
DURATION
- Exposure duration: 48 +/- 8 h
DETERMINATION OF CYTOTOXICITY
- Method: other: decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn. - Evaluation criteria:
- For a test article to be considered positive, it must produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.
Tester Strains TA1535, TA1537 and TA1538
For a test article to be considered positive, it must produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article. - Statistics:
- no data
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- - Precipitation: the test item formed a suspension in DMSO in the stock solution and all dilutions tested and precipitated at the highest concentration tested (5000 µg/plate)
RANGE-FINDING/SCREENING STUDIES:
The dose rangefinding study was conducted on the test article using tester strain TA100 in both the presence and absence of S9 (one plate per dose). Ten doses of test article, from 5,000 to 6.67 µg were tested. No cytotoxicity was observed in either the presence or absence of S9 as evidenced by no observed decrease in the number of revertants per plate. The bacterial background lawn was evaluated as normal up to the 1,000 pg per plate dose in both the presence and absence of S9. The lawns on the plates above this dose could not be evaluated due to the presence of test article precipitate. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
The test item (91235) did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay) with and without metabolic activation. - Executive summary:
Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 with (induced rat liver S9 mix) and without metabolic activation at concentrations of 100, 333, 667, 1000, 3330 and 5000 µg/plate using the plate incorporation assay. The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.
- 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:
- From 15 DEC 2004 to 10 JAN 2005
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD 471)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- in accordance to German Chemikaliengesetz and Directive 88/320/EEC
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- induced rat liver S9 (phenobarbital/ß-naphtoflavone used for induction)
- Test concentrations with justification for top dose:
- Experiment I (plate incorporation): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Experiment II (preincubation test): 33, 100, 333, 1000, 2500, and 5000 µg/plate - Vehicle / solvent:
- - Vehicle used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide (TA 1535, TA 100), 4-nitro-o-phenylene-diamine (TA1537, TA 98), methyl methan sulfonate (WP2uvrA)
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (all strains)
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Experiment I: plate incorporation
Experiment II: preincubation
DURATION
- Preincubation period: only Experiment II: 60 minutes at 37 °C
- Exposure duration: 48 hours
NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including controls - Evaluation criteria:
- test item considerer as a mutagen, if:
- number of revertants exceeding a threshold of twice (TA 98, TA 100 and WP2uvrA) or
- number of revertants exceeding a threshold of thrice (TA 1535, TA 1537)
the colony count of the corresponding solvent control
- a dose dependent increase is considered relevant if the threshold is exceeded more than once
- increase in threshold at only one concentration is judged relevant if reproduced in a second independent experiment
-dose dependent increase below the threshold is regarded as indication of a mutagenic potential if reproduced in a second independent experiment, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered relevant - Species / strain:
- other: S. typhimurium TA 1535, TA 98, TA 100 and TA 1537, E. coli WP2uvrA
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- minor effect in plate incorporation assay at 5000 µg/plate in TA1537 (reduction in the number of revertants to ~50% of control plates)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: visible in Experiment II without S9 at a concentration of 5000 µg/plate and with S9-Mix at concentrations of 1000 µg/plate and above
COMPARISON WITH HISTORICAL CONTROL DATA:
- historical range of positive controls was exceeded with metabolic activation in strains TA 98 and TA 1537 (Experiment I) and in strain TA 100 (Experiment/ and II), this effect indicates the sensitivity of the strains rather than compromising the assay
- Strain WP2uvrA (ExperimentI) with metabolic activation historical control range of negative and solvent controls was slightly exceeded
- Strain WP2uvrA and TA 1535 (ExperimentII) with and without metabolic activation historical control range of negative and solvent controls was slightly exceeded
- Strain TA 1535 and TA 98 (ExperimentII) with metabolic activation historical control range of negative and solvent controls was slightly exceeded
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- TA 1537: plate incorporation assay without metabolic activation minor effect at 5000 µg/plate - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and preincubation assay) with and without metabolic activation. - Executive summary:
Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA100 and Escherichia coli strain WP2uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate using the plate incorporation assay. Additionally, a preincubation assay with or without metabolic activation was performed using the concentrations 33, 100, 333, 1000, 2500, and 5000 µg/plate.
The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.
- 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:
- From 27 SEP 2004 to 13 OCT 2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD 471)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- in accordance to german Chemikaliengesetz and Directive 88/320/EEC
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- induced rat liver S9 (phenobarbital/ß-naphtoflavone used for induction)
- Test concentrations with justification for top dose:
- Experiment I (plate incorporation): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Experiment II (preincubation test): 33, 100, 333, 1000, 2500, and 5000 µg/plate - Vehicle / solvent:
- - Vehicle used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide (TA 1535, TA 100), 4-nitro-o-phenylene-diamine (TA1537, TA 98), methyl methan sulfonate (WP2uvrA)
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Experiment I: plate incorporation
Experiment II: preincubation
DURATION
- Preincubation period: only Experiment II: 60 minutes at 37 °C
- Exposure duration: 48 hours
NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including controls - Evaluation criteria:
- test item considerer as a mutagen, if:
- number of revertants exceeding a threshold of twice (TA 98, TA 100 and WP2uvrA) or
- number of revertants exceeding a threshold of thrice (TA 1535, TA 1537)
the colony count of the corresponding solvent control
- a dose dependent increase is considered relevant if the threshold is exceeded more than once
- increase in threshold at only one concentration is judged relevant if reproduced in a second independent experiment - Key result
- Species / strain:
- other: S. Typhimurium TA 98 and E.coli WP2uvrA
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative 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:
- cytotoxicity
- Remarks:
- minor effect in Experiment I without metabolic activation at a concentration of 2500 µg/plate and above (reduction in the number of revertants to ~50% of control plates)
- Vehicle controls validity:
- valid
- Untreated negative 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:
- cytotoxicity
- Remarks:
- minor effect in Experiment I without metabolic activation at a concentration of 5000 µg/plate and with metabolic activation at a concentration of 2500 µg/plate and above (reduction in the number of revertants to ~50% of control plates)
- Vehicle controls validity:
- valid
- Untreated negative 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:
- cytotoxicity
- Remarks:
- minor effect in Experiment I and II each with metabolic activation at a concentration of 5000 µg/plate (reduction in the number of revertants to ~50% of control plates)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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 with metabolic activation
negative without metabolic activation
The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and preincubation assay) with and without metabolic activation.
The findings of this study support the results given in the key study. - Executive summary:
Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA100 and Escherichia coli strain WP2uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate using the plate incorporation assay. Additionally, a preincubation assay with or without metabolic activation was performed using the concentrations 33, 100, 333, 1000, 2500, and 5000 µg/plate.
The test item did not reveal any mutagenic activity under the conditions tested.The appropriate reference mutagenes showed distinct positive mutagenic effects.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 20 January 2021 to 06 May 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- OECD 473
- Justification for type of information:
- See read across justification document in chapter 13
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted on 29th July 2016.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the solvent/vehicle: acetone - Target gene:
- Human Peripheral Blood Lymphocytes were used
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- Human Peripheral Blood Lymphocytes were used
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 homogenate
- Test concentrations with justification for top dose:
- 0.015625, 0.03125 and 0.0625 mg/mL, top dose selected based on initial cytotoxicity results
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: formed suspension in 200 mg/mL - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium - RPMI
DURATION
- Exposure duration: 3 to 6 hours and 24 hours
SELECTION AGENT (mutation assays): RPMI 1640 MEDIUM
STAIN : Giemsa
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Pellets were mixed with 4 to 5 mL of freshly prepared warm 0.56% Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 4 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cell suspension was incubated for 10 minutes at room temperature and later suspension was centrifuged at 2200 rpm for 10 minutes. The procedure was repeated twice by adding 4 mL of cold acetic acid: methanol fixative (1:3).
Clean slides were stored in a beaker with distilled water and kept in the refrigerator for at least 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension were aspirated and dropped onto the chilled slide pre labeled with study number, with (+S9) or without metabolic activation (-S9), treatment/group and slide number. The slides were air dried
NUMBER OF CELLS EVALUATED: 150 metaphase
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - Rationale for test conditions:
- • Three analyzable concentrations were used for chromosomal aberration test.
• Initial cytotoxicity was conducted at concentrations of 0.015625, 0.03125, 0.0625 and 0.125 mg/mL. The percentage reduction in Mitotic Index was in the range of 10.00 to 45.61 at 0.0625 mg/mL. As the percentage reduction in MI was not more than 45±5% at 0.0625 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.015625 and 0.03125 mg/mL - Evaluation criteria:
- Percentage mitotic index
- Statistics:
- yes
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change in pH was observed in any of the concentrations tested upto 2 mg/mL
- Water solubility: insoluble
- Precipitation: Precipitation test was conducted at 0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 24 hours of incubation, heavy precipitation was observed at 0.5, 1 and 2 mg/mL, moderate precipitation was observed at 0.125, 0.25 mg/mL and mild precipitation was observed at 0.0625 mg/mL. No precipitation was observed at 0.03125 and 0.015625 mg/mL
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: NA
- Negative (solvent/vehicle) historical control data: NEGATIVE CONTROLS MAINTAINED. - Conclusions:
- Based on the results obtained, the test item is considered as non-clastogenic upto the concentration of 0.0625 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 suspension in acetone at 200 mg/mL. Precipitation and pH test was conducted at 0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 24 hours of incubation, heavy precipitation was observed at 0.5, 1 and 2 mg/mL, moderate precipitation was observed at 0.25, 0.125 mg/mL, mild precipitation was observed at 0.625 mg/mL. No precipitation was observed at 0.03125 and 0.015625 mg/mL. No change in pH was observed in any of the concentrations tested upto 2 mg/mL. Hence, 0.125 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.015625, 0.03125 and 0.0625 mg/mL of test item.
In initial cytotoxicity test, the percentage reduction in Mitotic Index was in the range of 10.00 to 45.61 at 0.015625, 0.03125 and 0.0625mg/mL (as there were colored particles and precipitation was interfering with the cells, the slides could not be evaluated at 0.125 mg/mL) in presence of metabolic activation (short term), in absence of metabolic activation (short term) in absence of metabolic activation (long term), respectively. As the percentage reduction inmitotic index was not more than 45±5% at 0.0625 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. other concentrations tested were 0.015625 and 0.03125 mg/mL.
In the chromosomal aberration test, the cells were treated with test item at the concentrations of 0.015625, 0.03125 and 0.0625mg/mLusing acetone 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.
Theconcurrent vehicle control values were within the 95% control limits of the distribution of the laboratory’s historical vehicle control database.
- Endpoint:
- genetic toxicity in vitro, other
- Remarks:
- Mammalian Cell Gene Mutation Tests
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 02 July 2021 to 04 November 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- See read across justification document in chapter 13
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted on 29th July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes
- Target gene:
- CHO AA8 cells
- Species / strain / cell type:
- other: CHO AA8 cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: American Type Culture Collection (ATCC)
- Cell doubling time : Approximately 12 hours
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Alpha MEM CULTURE MEDIA with 10% FBS and 1% penstrep and 5±1% CO2
- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Doubling time model chromosome number cells were checked. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 homogenate
- Test concentrations with justification for top dose:
- 15.625, 31.25, 62.5 and 125 µg/mL based on initial cytotoxicity test
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- benzo(a)pyrene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 2×107
DURATION
- Exposure duration: 3 hours and 38 minutes
- Expression time (cells in growth medium): 7 days for mutant phenotype expression
- Selection time (if incubation with a selection agent):8 days
SELECTION AGENT (mutation assays): 10 µM of 6-Thioguanine
STAIN : 5% giemsa
NUMBER OF REPLICATIONS: 5
METHODS STAINING TECHNIQUE USED: Post incubation period, medium from each culture flask was aspirated and stained with 5% Giemsa stain. Number of colonies formed was counted manually.
DETERMINATION OF CYTOTOXICITY
- Method:cloning efficiency - Rationale for test conditions:
- Acceptance of a test is based on the following criteria:
•The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database as described in OECD guidelines for testing of chemicals, No. 476.
•Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control.
•Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
•Adequate number of cells and concentrations are analysable (according to OECD guidelines for testing of chemicals, No. 476).
•The criteria for the selection of top concentration are consistent with those described in OECD guidelines for testing of chemicals, No. 476. - Evaluation criteria:
- Colony counts in selective media and non-selective media
- Statistics:
- yes, Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0.
- Key result
- Species / strain:
- other: CHO AA8 cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change in pH was observed in any of the test concentrations
- Water solubility: Insoluble
- Precipitation: The precipitation and pH were tested at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL concentrations. Post 3 hours of incubation, no precipitation was observed at the tested concentrations of 0.03125, 0.0625 mg/mL, moderate precipitation was observed at 0.125 and 0.25 mg/mL, heavy precipitation was observed at 0.5, 1 and 2 mg/mL
RANGE-FINDING/SCREENING STUDIES: Yes
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Positive Control- Benzo(a)pyrene and 4- Nitroquinoline N-oxide
With Metabolic Activation
(3 to 6 hours)
[Benzo(a)pyrene] Without Metabolic Activation
(3 to 6 hours)
[4 Nitroquinoline N-oxide]
Mean Data of Mutant Frequency/2x106 Cells 261.94 264.60
Standard
Deviation 27.28 18.52
Margin of Error 17.82 12.10
Upper bound 279.76 276.70
Lower bound 244.12 252.50
- Negative (solvent/vehicle) historical control data: Negative controls were maintained.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Relative survivality - Conclusions:
- The test item is considered as non-mutagenic at and up to the concentration of 125 µg/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 formed a suspension in acetone at concentration of 200 mg/mL. After 3 hours of incubation, no precipitation was observed at the tested concentrations of 0.03125 and 0.0625 mg/mL, moderate precipitation was observed at 0.125 and 0.25 mg/mL, and heavy precipitation was observed at 0.5, 1 and 2 mg/mL. No change in pH was observed in any of the test concentrations.
On the basis of precipitation results, 0.125 mg/mL (125 µg/mL) was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 7.8125, 15.625, 31.25, 62.5 and 125 µg/mL using acetone as a vehicle in four flasks/group in the presence and absence of metabolic activation (4 hours and 5 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% (62.61 % in presence of metabolic activation and64.60 % in absence of metabolic activation) at 125 µg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 125 µg/mL was selected as highest concentration for gene mutation test.
The gene mutation test was conducted at the concentrations of 125, 62.5, 31.25 and 15.625 µg/mL using acetone as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 38 minutes).Benzo(a) pyrene and4 Nitroquinoline N-oxidewere used aspositive controlsfor the gene mutation test.
Cytotoxicity as Relative Survival was 64.10 % in presence of metabolic activation and63.48 % in absence of metabolic activationat the highest tested concentration of 125µg/mL. Relative Survival of negative control was 100.00 in presence of metabolic activation and 103.48 in absence of metabolic activation.There was no statistically significant increase in mutant frequencies at any of the concentrations tested and negative control when compared with the vehicle control.
There was statistically significant increase in mutant frequenciesfor positive controlswhen compared with the vehicle controlin bothmetabolic activation andabsence of metabolic activation.
Referenceopen allclose all
Experiment without metabolic activation:
Treatments at 3500 µg/m1 and 5000 µg/ml were moderately cytotoxic (29.3% and 31.1% relative growths). The remaining four dose levels were noncytotoxic (83.3% to 105.8% relative growths). In order for a culture to be evaluated as mutagenic, a mutant frequency greater than 110.1 x 10-6was required. This threshold value was equal to twice the average mutant frequency of the concurrent vehicle controls. None of the analyzed treatments induced a mutant frequency that exceeded the minimum criterion and no dose-related trend was observed.
Experiment with metabolic activation:
Treatments at 313 µg/ml and 2500 µg/m1 were terminated because sufficient noncytotoxic treatments were available for analysis. Concentrations of ¿3500 µg/ml induced weak cytotoxicity (down to 55.3% relative growth). None of the six analyzed treatments induced a mutant frequency that exceeded the minimum criterion of 88.6 x 10-6and no dose-related response was observed.
The results of the initial assay were confirmed by an independent confirmatory assay.
TABLE 1. SUMMARY OF INITIAL CYTOTOXICITY TEST
Set No. | Treatment | Concentration (µg/mL) | Average Colony Count± SD | Cloning Efficiency (CE) | Adjusted Cloning Efficiency (ACE) | Relative Survival (RS) (%) | ||
Set 1 +S9 | Vehicle Control (Acetone) | - | 189.67 | ± | 2.08 | 0.95 | 1.15 | - |
test item | 7.8125 | 185.67 | ± | 1.53 | 0.93 | 1.10 | 95.65 | |
15.625 | 180.67 | ± | 5.03 | 0.90 | 1.06 | 92.17 | ||
31.25 | 175.33 | ± | 3.51 | 0.88 | 1.00 | 86.96 | ||
62.5 | 164.33 | ± | 4.04 | 0.82 | 0.88 | 76.52 | ||
125 | 144.00 | ± | 5.20 | 0.72 | 0.72 | 62.61 | ||
Set 2 -S9 | Vehicle Control (Acetone) | - | 187.67 | ± | 2.52 | 0.94 | 1.13 | - |
test item | 7.8125 | 182.33 | ± | 2.52 | 0.91 | 1.06 | 93.81 | |
15.625 | 182.00 | ± | 3.00 | 0.91 | 1.04 | 92.04 | ||
31.25 | 178.00 | ± | 2.65 | 0.89 | 0.99 | 87.61 | ||
62.5 | 165.00 | ± | 4.58 | 0.83 | 0.90 | 79.65 | ||
125 | 142.67 | ± | 9.45 | 0.71 | 0.73 | 64.60 | ||
+S9: with metabolic activation; -S9: without metabolic activation;
Adjusted CE = CE × Number of cells at the end of treatment/number of cells at the beginning of treatment.
RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.
CE = Number of colonies/Number of cells plated.
TABLE 1. SUMMARY OF PARALLEL CYTOTOXICITY TEST- GENE MUTATION TEST
Set No. | Treatment | Concentration (µg/mL) | Average Colony Count ± SD | Cloning Efficiency (CE) | Adjusted Cloning Efficiency (ACE) | Relative Survival (RS) (%) | ||
Set 1 +S9 | Vehicle Control (Acetone) | - | 190.33 | ± | 1.53 | 0.95 | 1.17 | - |
Negative control | - | 190.67 | ± | 1.15 | 0.95 | 1.17 | 100.00 | |
test item | 15.625 | 182.00 | ± | 2.00 | 0.91 | 1.09 | 93.16 | |
31.25 | 172.67 | ± | 4.51 | 0.86 | 0.99 | 84.62 | ||
62.5 | 168.33 | ± | 2.08 | 0.84 | 0.90 | 76.92 | ||
125 | 147.67 | ± | 2.52 | 0.74 | 0.75 | 64.10 | ||
Benzo(a)pyrene (Positive Control) | 3 | 180.00 | ± | 7.81 | 0.90 | 1.07 | 91.45 | |
Set 2 | Vehicle Control (Acetone) | - | 188.67 | ± | 5.03 | 0.94 | 1.15 | - |
Negative control | - | 189.33 | ± | 2.08 | 0.95 | 1.19 | 103.48 | |
test item | 15.625 | 178.67 | ± | 1.15 | 0.89 | 1.07 | 93.04 | |
31.25 | 174.33 | ± | 5.03 | 0.87 | 1.01 | 87.83 | ||
62.5 | 167.33 | ± | 5.51 | 0.84 | 0.88 | 76.52 | ||
125 | 143.00 | ± | 4.58 | 0.72 | 0.73 | 63.48 | ||
4 Nitroquinoline N-oxide (Positive Control) | 1 | 179.33 | ± | 4.51 | 0.90 | 1.09 | 94.78 | |
+S9: with metabolic activation; -S9: without metabolic activation;
*Note: Cloning Efficiency = 200 cells plated for each replicate.
RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.
CE = Number of colonies/Number of cells plated.
Adjusted CE = CE × Number of cells at the end of treatment/number of cells at the beginning of treatment.
Set No. | Treatment | Concentration (µg/mL) | *Average Colony Count ± SD | Cloning Efficiency in selective media | Cloning Efficiency in non-selective media* | Total number of Mutant Colonies/ 2×106cells | Mutant Frequency/ 2×106cells | ||
Set 1 +S9 | Vehicle Control (Acetone) | - | 186.67 | ± | 4.04 | 0.0000105 | 0.93 | 21 | 22.58 |
Negative control | - | 187.00 | ± | 2.65 | 0.0000110 | 0.94 | 22 | 23.40 | |
15.625 | 186.00 | ± | 1.00 | 0.0000110 | 0.93 | 22 | 23.66 | ||
31.25 | 183.33 | ± | 3.79 | 0.0000110 | 0.92 | 22 | 23.91 | ||
62.5 | 178.67 | ± | 2.31 | 0.0000110 | 0.89 | 22 | 24.72 | ||
125 | 175.67 | ± | 3.51 | 0.0000105 | 0.88 | 21 | 23.86 | ||
Benzo(a)pyrene (Positive Control) | 3 | 181.67 | ± | 1.53 | 0.0001140 | 0.91 | 228 | 250.55** | |
Set 2 -S9 | Vehicle Control (Acetone) | - | 188.33 | ± | 2.08 | 0.0000115 | 0.94 | 23 | 24.47 |
Negative control | - | 187.33 | ± | 4.04 | 0.0000115 | 0.94 | 23 | 24.47 | |
test item | 15.625 | 184.67 | ± | 5.51 | 0.0000115 | 0.92 | 23 | 25.00 | |
31.25 | 183.00 | ± | 5.57 | 0.0000120 | 0.92 | 24 | 26.09 | ||
62.5 | 182.33 | ± | 6.03 | 0.0000110 | 0.91 | 22 | 24.18 | ||
125 | 174.33 | ± | 5.03 | 0.0000115 | 0.87 | 23 | 26.44 | ||
4 Nitroquinoline N-oxide (Positive Control) | 1 | 183.33 | ± | 4.51 | 0.0001170 | 0.92 | 234 | 254.35** | |
TABLE 1. SUMMARY OF GENE MUTATION TEST
+S9: with metabolic activation; -S9: without metabolic activation.
*Note: Cloning efficiency = 200 cells plated for each replicate.
**: Statistically significant (p˂0.05).
Mutant Frequency = Cloning efficiency of mutant colonies in selective medium/Cloning efficiency in non- selective medium.
Pre study:
TABLE 1. SUMMARY OF PERCENTAGE MITOTIC INDEX FOR INITIAL CYTOTOXICITY TEST
Set No. | Treatment | Concentrations (mg/mL) | Average % Mitotic Index | % Reduction of Mitotic Index | ||||||
Set 1 (+S9) (3 to 6 hours) | Vehicle Control | 0 | 7.50 | - | ||||||
Test item | NC | 7.27 | 3.07 | |||||||
0.015625 | 6.75 | 10.00 | ||||||||
0.03125 | 5.99 | 20.13 | ||||||||
0.0625 | 4.30 | 42.67 | ||||||||
0.125 | - | - | ||||||||
Set 2 (-S9) (3 to 6 hours) | Vehicle Control | 0 | 7.51 | - | ||||||
Test item | NC | 7.17 | 4.53 | |||||||
0.015625 | 6.74 | 10.25 | ||||||||
0.03125 | 6.11 | 18.64 | ||||||||
0.0625 | 4.34 | 42.21 | ||||||||
0.125 | - | - | ||||||||
Set 3 (-S9) (20 to 24 hours) | Vehicle Control | 0 | 7.85 | - | ||||||
Test item | NC | 7.52 | 4.20 | |||||||
0.015625 | 6.69 | 14.78 | ||||||||
0.03125 | 6.03 | 23.18 | ||||||||
0.0625 | 4.27 | 45.61 | ||||||||
0.125 | - | - |
+S9: With metabolic activation, -S9: Without metabolic activation.
Main study :
TABLE 1. SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX
Set No. | Treatment | Concentrations (mg/mL) | Mean % MI | Mean % Reduction in MI | Mean of Total Aberrations with Gaps | Mean of Total Aberrations without Gaps | Mean of Total Aberrant cells without Gaps | Mean of Percentage Aberrated Cells |
Set 1 (+S9) (3 to 6 hours) | Vehicle Control | 0 | 7.65 | NA | 2.0 | 2.0 | 2.0 | 1.33 |
Positive Control (Cyclophosphamide monohydrate) | 10 (µg/mL) | 7.26 | 5.1 | 17.0 | 17.0 | 15.5 | 10.34* | |
Test item | NC | 7.36 | 3.79 | 2.0 | 2.0 | 1.5 | 1.00 | |
0.015625 | 6.47 | 15.42 | 1.5 | 1.5 | 1.5 | 1.00 | ||
0.03125 | 5.85 | 23.53 | 2.5 | 2.5 | 2.0 | 1.33 | ||
0.0625 | 4.16 | 45.62 | 2.5 | 2.5 | 2.5 | 1.67 |
MI: Mitotic Index; *: Statistically significant; +S9: With metabolic activation.
TABLE 2 (Contd…). SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX
Refer Appendix 2
Set No. | Treatment | Concentrations (mg/mL) | Mean % MI | Mean % Reduction in MI | Mean of Total Aberrations with Gaps | Mean of Total Aberrations without Gaps | Mean of Total Aberrant cells without Gaps | Mean of Percentage Aberrated Cells |
Set 2 (-S9) (3 to 6 hours) | Vehicle Control | 0 | 7.67 | NA | 2 | 2 | 2 | 1.33 |
Positive Control (Mitomycin-C) | 0.05 (µg/mL) | 6.97 | 9.13 | 18.5 | 18 | 15 | 10.00* | |
Test item | NC | 7.29 | 4.95 | 1.5 | 1.5 | 1.5 | 1.00 | |
0.015625 | 6.72 | 12.39 | 1.5 | 1.5 | 1.5 | 1.00 | ||
0.03125 | 6.24 | 18.64 | 2.2 | 2.5 | 2 | 1.33 | ||
0.0625 | 4.44 | 42.11 | 2.5 | 2.5 | 2 | 1.33 |
MI: Mitotic Index; *: Statistically significant; -S9: Without metabolic activation.
TABLE 2 (Contd…).SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX
Set No. | Treatment | Concentrations (mg/mL) | Mean % MI | Mean % Reduction in MI | Mean of Total Aberrations with Gaps | Mean of Total Aberrations without Gaps | Mean of Total Aberrant cells without Gaps | Mean of Percentage Aberrated Cells |
Set 3 (-S9) (20 to 24 hours) | Vehicle Control | 0 | 7.81 | NA | 2.00 | 2.00 | 2.00 | 1.33 |
Positive Control (Mitomycin-C) | 0.05 (µg/mL) | 7.17 | 8.19 | 19.50 | 19.00 | 16.00 | 10.67* | |
Test item | NC | 7.41 | 5.12 | 2.50 | 2.50 | 2.00 | 1.33 | |
0.015625 | 6.50 | 16.77 | 1.50 | 1.50 | 1.50 | 1.00 | ||
0.03125 | 5.84 | 25.22 | 2.50 | 2.50 | 2.00 | 1.33 | ||
0.0625 | 4.220 | 45.97 | 3 | 3 | 2.5 | 1.67 |
MI: Mitotic Index; *: Statistically significant; -S9: Without metabolic activation.
Experiment without metabolic activation:
Treatments at 3500 µg/m1 and 5000 µg/ml were moderately cytotoxic (29.3% and 31.1% relative growths). The remaining four dose levels were noncytotoxic (83.3% to 105.8% relative growths). In order for a culture to be evaluated as mutagenic, a mutant frequency greater than 110.1 x 10-6was required. This threshold value was equal to twice the average mutant frequency of the concurrent vehicle controls. None of the analyzed treatments induced a mutant frequency that exceeded the minimum criterion and no dose-related trend was observed.
Experiment with metabolic activation:
Treatments at 313 µg/ml and 2500 µg/m1 were terminated because sufficient noncytotoxic treatments were available for analysis. Concentrations of ¿3500 µg/ml induced weak cytotoxicity (down to 55.3% relative growth). None of the six analyzed treatments induced a mutant frequency that exceeded the minimum criterion of 88.6 x 10-6and no dose-related response was observed.
The results of the initial assay were confirmed by an independent confirmatory assay.
The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.
The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.
Pre study:
TABLE 1. SUMMARY OF PERCENTAGE MITOTIC INDEX FOR INITIAL CYTOTOXICITY TEST
Set No. | Treatment | Concentrations (mg/mL) | Average % Mitotic Index | % Reduction of Mitotic Index | ||||||
Set 1 (+S9) (3 to 6 hours) | Vehicle Control | 0 | 7.50 | - | ||||||
Test item | NC | 7.27 | 3.07 | |||||||
0.015625 | 6.75 | 10.00 | ||||||||
0.03125 | 5.99 | 20.13 | ||||||||
0.0625 | 4.30 | 42.67 | ||||||||
0.125 | - | - | ||||||||
Set 2 (-S9) (3 to 6 hours) | Vehicle Control | 0 | 7.51 | - | ||||||
Test item | NC | 7.17 | 4.53 | |||||||
0.015625 | 6.74 | 10.25 | ||||||||
0.03125 | 6.11 | 18.64 | ||||||||
0.0625 | 4.34 | 42.21 | ||||||||
0.125 | - | - | ||||||||
Set 3 (-S9) (20 to 24 hours) | Vehicle Control | 0 | 7.85 | - | ||||||
Test item | NC | 7.52 | 4.20 | |||||||
0.015625 | 6.69 | 14.78 | ||||||||
0.03125 | 6.03 | 23.18 | ||||||||
0.0625 | 4.27 | 45.61 | ||||||||
0.125 | - | - |
+S9: With metabolic activation, -S9: Without metabolic activation.
Main study :
TABLE 1. SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX
Set No. | Treatment | Concentrations (mg/mL) | Mean % MI | Mean % Reduction in MI | Mean of Total Aberrations with Gaps | Mean of Total Aberrations without Gaps | Mean of Total Aberrant cells without Gaps | Mean of Percentage Aberrated Cells |
Set 1 (+S9) (3 to 6 hours) | Vehicle Control | 0 | 7.65 | NA | 2.0 | 2.0 | 2.0 | 1.33 |
Positive Control (Cyclophosphamide monohydrate) | 10 (µg/mL) | 7.26 | 5.1 | 17.0 | 17.0 | 15.5 | 10.34* | |
Test item | NC | 7.36 | 3.79 | 2.0 | 2.0 | 1.5 | 1.00 | |
0.015625 | 6.47 | 15.42 | 1.5 | 1.5 | 1.5 | 1.00 | ||
0.03125 | 5.85 | 23.53 | 2.5 | 2.5 | 2.0 | 1.33 | ||
0.0625 | 4.16 | 45.62 | 2.5 | 2.5 | 2.5 | 1.67 |
MI: Mitotic Index; *: Statistically significant; +S9: With metabolic activation.
TABLE 2 (Contd…). SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX
Refer Appendix 2
Set No. | Treatment | Concentrations (mg/mL) | Mean % MI | Mean % Reduction in MI | Mean of Total Aberrations with Gaps | Mean of Total Aberrations without Gaps | Mean of Total Aberrant cells without Gaps | Mean of Percentage Aberrated Cells |
Set 2 (-S9) (3 to 6 hours) | Vehicle Control | 0 | 7.67 | NA | 2 | 2 | 2 | 1.33 |
Positive Control (Mitomycin-C) | 0.05 (µg/mL) | 6.97 | 9.13 | 18.5 | 18 | 15 | 10.00* | |
Test item | NC | 7.29 | 4.95 | 1.5 | 1.5 | 1.5 | 1.00 | |
0.015625 | 6.72 | 12.39 | 1.5 | 1.5 | 1.5 | 1.00 | ||
0.03125 | 6.24 | 18.64 | 2.2 | 2.5 | 2 | 1.33 | ||
0.0625 | 4.44 | 42.11 | 2.5 | 2.5 | 2 | 1.33 |
MI: Mitotic Index; *: Statistically significant; -S9: Without metabolic activation.
TABLE 2 (Contd…).SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX
Set No. | Treatment | Concentrations (mg/mL) | Mean % MI | Mean % Reduction in MI | Mean of Total Aberrations with Gaps | Mean of Total Aberrations without Gaps | Mean of Total Aberrant cells without Gaps | Mean of Percentage Aberrated Cells |
Set 3 (-S9) (20 to 24 hours) | Vehicle Control | 0 | 7.81 | NA | 2.00 | 2.00 | 2.00 | 1.33 |
Positive Control (Mitomycin-C) | 0.05 (µg/mL) | 7.17 | 8.19 | 19.50 | 19.00 | 16.00 | 10.67* | |
Test item | NC | 7.41 | 5.12 | 2.50 | 2.50 | 2.00 | 1.33 | |
0.015625 | 6.50 | 16.77 | 1.50 | 1.50 | 1.50 | 1.00 | ||
0.03125 | 5.84 | 25.22 | 2.50 | 2.50 | 2.00 | 1.33 | ||
0.0625 | 4.220 | 45.97 | 3 | 3 | 2.5 | 1.67 |
MI: Mitotic Index; *: Statistically significant; -S9: Without metabolic activation.
TABLE 1. SUMMARY OF INITIAL CYTOTOXICITY TEST
Set No. | Treatment | Concentration (µg/mL) | Average Colony Count± SD | Cloning Efficiency (CE) | Adjusted Cloning Efficiency (ACE) | Relative Survival (RS) (%) | ||
Set 1 +S9 | Vehicle Control (Acetone) | - | 189.67 | ± | 2.08 | 0.95 | 1.15 | - |
test item | 7.8125 | 185.67 | ± | 1.53 | 0.93 | 1.10 | 95.65 | |
15.625 | 180.67 | ± | 5.03 | 0.90 | 1.06 | 92.17 | ||
31.25 | 175.33 | ± | 3.51 | 0.88 | 1.00 | 86.96 | ||
62.5 | 164.33 | ± | 4.04 | 0.82 | 0.88 | 76.52 | ||
125 | 144.00 | ± | 5.20 | 0.72 | 0.72 | 62.61 | ||
Set 2 -S9 | Vehicle Control (Acetone) | - | 187.67 | ± | 2.52 | 0.94 | 1.13 | - |
test item | 7.8125 | 182.33 | ± | 2.52 | 0.91 | 1.06 | 93.81 | |
15.625 | 182.00 | ± | 3.00 | 0.91 | 1.04 | 92.04 | ||
31.25 | 178.00 | ± | 2.65 | 0.89 | 0.99 | 87.61 | ||
62.5 | 165.00 | ± | 4.58 | 0.83 | 0.90 | 79.65 | ||
125 | 142.67 | ± | 9.45 | 0.71 | 0.73 | 64.60 | ||
+S9: with metabolic activation; -S9: without metabolic activation;
Adjusted CE = CE × Number of cells at the end of treatment/number of cells at the beginning of treatment.
RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.
CE = Number of colonies/Number of cells plated.
TABLE 1. SUMMARY OF PARALLEL CYTOTOXICITY TEST- GENE MUTATION TEST
Set No. | Treatment | Concentration (µg/mL) | Average Colony Count ± SD | Cloning Efficiency (CE) | Adjusted Cloning Efficiency (ACE) | Relative Survival (RS) (%) | ||
Set 1 +S9 | Vehicle Control (Acetone) | - | 190.33 | ± | 1.53 | 0.95 | 1.17 | - |
Negative control | - | 190.67 | ± | 1.15 | 0.95 | 1.17 | 100.00 | |
test item | 15.625 | 182.00 | ± | 2.00 | 0.91 | 1.09 | 93.16 | |
31.25 | 172.67 | ± | 4.51 | 0.86 | 0.99 | 84.62 | ||
62.5 | 168.33 | ± | 2.08 | 0.84 | 0.90 | 76.92 | ||
125 | 147.67 | ± | 2.52 | 0.74 | 0.75 | 64.10 | ||
Benzo(a)pyrene (Positive Control) | 3 | 180.00 | ± | 7.81 | 0.90 | 1.07 | 91.45 | |
Set 2 | Vehicle Control (Acetone) | - | 188.67 | ± | 5.03 | 0.94 | 1.15 | - |
Negative control | - | 189.33 | ± | 2.08 | 0.95 | 1.19 | 103.48 | |
test item | 15.625 | 178.67 | ± | 1.15 | 0.89 | 1.07 | 93.04 | |
31.25 | 174.33 | ± | 5.03 | 0.87 | 1.01 | 87.83 | ||
62.5 | 167.33 | ± | 5.51 | 0.84 | 0.88 | 76.52 | ||
125 | 143.00 | ± | 4.58 | 0.72 | 0.73 | 63.48 | ||
4 Nitroquinoline N-oxide (Positive Control) | 1 | 179.33 | ± | 4.51 | 0.90 | 1.09 | 94.78 | |
+S9: with metabolic activation; -S9: without metabolic activation;
*Note: Cloning Efficiency = 200 cells plated for each replicate.
RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.
CE = Number of colonies/Number of cells plated.
Adjusted CE = CE × Number of cells at the end of treatment/number of cells at the beginning of treatment.
Set No. | Treatment | Concentration (µg/mL) | *Average Colony Count ± SD | Cloning Efficiency in selective media | Cloning Efficiency in non-selective media* | Total number of Mutant Colonies/ 2×106cells | Mutant Frequency/ 2×106cells | ||
Set 1 +S9 | Vehicle Control (Acetone) | - | 186.67 | ± | 4.04 | 0.0000105 | 0.93 | 21 | 22.58 |
Negative control | - | 187.00 | ± | 2.65 | 0.0000110 | 0.94 | 22 | 23.40 | |
15.625 | 186.00 | ± | 1.00 | 0.0000110 | 0.93 | 22 | 23.66 | ||
31.25 | 183.33 | ± | 3.79 | 0.0000110 | 0.92 | 22 | 23.91 | ||
62.5 | 178.67 | ± | 2.31 | 0.0000110 | 0.89 | 22 | 24.72 | ||
125 | 175.67 | ± | 3.51 | 0.0000105 | 0.88 | 21 | 23.86 | ||
Benzo(a)pyrene (Positive Control) | 3 | 181.67 | ± | 1.53 | 0.0001140 | 0.91 | 228 | 250.55** | |
Set 2 -S9 | Vehicle Control (Acetone) | - | 188.33 | ± | 2.08 | 0.0000115 | 0.94 | 23 | 24.47 |
Negative control | - | 187.33 | ± | 4.04 | 0.0000115 | 0.94 | 23 | 24.47 | |
test item | 15.625 | 184.67 | ± | 5.51 | 0.0000115 | 0.92 | 23 | 25.00 | |
31.25 | 183.00 | ± | 5.57 | 0.0000120 | 0.92 | 24 | 26.09 | ||
62.5 | 182.33 | ± | 6.03 | 0.0000110 | 0.91 | 22 | 24.18 | ||
125 | 174.33 | ± | 5.03 | 0.0000115 | 0.87 | 23 | 26.44 | ||
4 Nitroquinoline N-oxide (Positive Control) | 1 | 183.33 | ± | 4.51 | 0.0001170 | 0.92 | 234 | 254.35** | |
TABLE 1. SUMMARY OF GENE MUTATION TEST
+S9: with metabolic activation; -S9: without metabolic activation.
*Note: Cloning efficiency = 200 cells plated for each replicate.
**: Statistically significant (p˂0.05).
Mutant Frequency = Cloning efficiency of mutant colonies in selective medium/Cloning efficiency in non- selective medium.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
in-vivo:
Pigment red 202 (not specified form): negative (MN)
Structure analogue: Pigment red 122 (nano form): negative (MN)
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 28 EFEB 1995 to 15 MAR 1995
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: comparable to guideline study (OECD 474) with some restrictions (insufficient number of PCE examined, but two doses above limit dose tested)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- : insufficient number of polychromatic erythrocytes examined
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI, USA
- Age at study initiation: about 9 weeks
- Weight at study initiation: males: 31.0-38.5 g; females: 23.1-30.3 g
- Assigned to test groups randomly: yes
- Housing: seven per cage during quarantine, up to 5 per cage at randomisation
- Diet (ad libitum): Purina Certified Laboratory Chow No. 5002
- Water: ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 +/- 3.3
- Humidity (%): 55 +/- 15
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle used: corn oil
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle: stock solution 250 mg/ml - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: stock solution: 8.75 g test item in corn oil, 35 ml final volume yielded an opaque, red suspension with a concentration of 250 mg/ml
Constant dosing volume: 20 ml/kg - Duration of treatment / exposure:
- up to 72 h
- Frequency of treatment:
- single oral exposure
- Post exposure period:
- 24, 48 and 72 h
- Remarks:
- Doses / Concentrations:
1250, 2500, 5000 mg/kg
Basis:
nominal conc. - No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide, 80 mg/kg
- Tissues and cell types examined:
- erythrocytes from bone marrow
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
based on a range finding pre-test (no mortality or clinical symptoms of toxicity after administration of doses up to 5000 mg/kg)
DETAILS OF SLIDE PREPARATION:
At the appropriate harvest time, the animals were euthanatized with CO2 and the adhering soft tissue and epiphyses of both tibiae were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 3-5 ml bovine serum (one tube for each animal). Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. The slides were fixed in methanol and stained in May-Grunwald solution followed by Giemsa. The air-dried slides were coverslipped using Depex® mounting medium.
METHOD OF ANALYSIS:
The coded slides were then scored for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is about 0.0-0.4%.
The frequency of PCEs versus NCEs was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring the first 1000 erythrocytes.
- Evaluation criteria:
- The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant positive response for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based an scientific judgment.
- Statistics:
- The analysis of the data was performed using an Analysis of Variance on the square root arcsine transformation which was performed on the proportion of cells with micronuclei per animal (square root arcsine proportion). Once the Analysis of Variance had been performed, Tukey's Studentized range test (HSD) with adjustment for multiple comparisons was used at each harvest time to determine which dose groups, if any, were significantly different (p<0.05) from the vehicle control. Analyses were performed separately for each harvest time and sex combination, and also at each harvest time for the sexes combined.
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): significantly less in the 2500 mg/kg group (males, 24 h harvest) and 5000 mg/kg group (females, 24 and 72 h harvest) compared to control group
- Appropriateness of dose levels and route: yes - Conclusions:
- Interpretation of results (migrated information): negative
The test item 2,9-dichloroquinacridone did not show mutagenic activity in the mouse micronucleus assay in vivo. - Executive summary:
In this mouse micronucleus assay, the test item was suspended in corn oil and applied to CD-1 mice by oral gavage at 1250, 2500, and 5000 mg/kg, based upon results of a range finding pre-test. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. The animals dosed with the test article were euthanatized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow. Vehicle and positive control groups, euthanatized approximately 24 hours after dosing, were included in the assay.
The test item did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay. The positive control group showed a significant positive response.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).
4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13). - Reason / purpose for cross-reference:
- read-across source
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results (migrated information): negative
The test item 2,9-dichloroquinacridone did not show mutagenic activity in the mouse micronucleus assay in vivo. - Executive summary:
In this mouse micronucleus assay, the test item was suspended in corn oil and applied to CD-1 mice by oral gavage at 1250, 2500, and 5000 mg/kg, based upon results of a range finding pre-test. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. The animals dosed with the test article were euthanatized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow. Vehicle and positive control groups, euthanatized approximately 24 hours after dosing, were included in the assay.
The test item did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay. The positive control group showed a significant positive response.
Referenceopen allclose all
All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest times. All test article dosed groups appeared normal immediately after dosing. Approximately 23, 47, and 71 hours after dosing, all animals appeared normal, but had diluted pink hair coats due to grooming and compound colored feces. All animals remained healthy until the appropriate harvest times.
The test item induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. Possibly due to toxicity induced by 2,9-Dichloroquinacridone, the PCE/NCE ratios of the 24 and 72 hour high dose females, the 24 hour males dosed with 2500 mg/kg were significantly less than the vehicle control group. The PCE/NCE ratio of the CP-treated females was also significantly less than the vehicle control group. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 2.40% ± 0.24% and 2.42% ± 0.36% for the males and females, respectively.
Treatment |
Dose |
Harvest time (h) |
% Micronucleated PCEs, |
Ration PCe/NCE |
|||
|
|
|
males |
females |
total |
males |
females |
Vehicle (corn oil) |
20 ml/kg |
24 |
0.02 ± 0.02 |
0.06 ± 0.02 |
0.04 ± 0.02 |
0.55 ± 0.07 |
1.03 ± 0.07 |
Cyclophosphamide |
80 mg/kg |
24 |
2.40 ± 0.24* |
2.42 ± 0.36* |
2.41 ± 0.20* |
0.59 ± 0.02 |
0.65 ± 0.05* |
Test item |
1250 mg/kg |
24 |
0.04 ± 0.02 |
0.06 ± 0.02 |
0.05 ± 0.02 |
0.33 ± 0.04 |
0.96 ± 0.07 |
|
48 |
0.04 ± 0.02 |
0.00 ± 0.00 |
0.02 ± 0.01 |
0.62 ± 0.06 |
0.73 ± 0.10 |
|
|
72 |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.65 ± 0.08 |
0.64 ± 0.14 |
|
|
2500 mg/kg |
24 |
0.02 ± 0.02 |
0.08 ± 0.06 |
0.05 ± 0.03 |
0.28 ± 0.04* |
0.89 ± 0.16 |
|
48 |
0.06 ± 0.02 |
0.00 ± 0.00 |
0.03 ± 0.02 |
0.49 ± 0.06 |
0.72 ± 0.15 |
|
|
72 |
0.08 ± 0.02 |
0.08 ± 0.04 |
0.08 ± 0.02 |
0.69 ± 0.02 |
0.69 ± 0.10 |
|
|
5000 mg/kg |
24 |
0.02 ± 0.02 |
0.04 ± 0.02 |
0.03 ± 0.02 |
0.49 ± 0.02 |
0.64 ± 0.10* |
|
48 |
0.06 ± 0.02 |
0.02 ± 0.02 |
0.04 ± 0.02 |
0.55 ± 0.11 |
0.80 ± 0.12 |
|
|
72 |
0.02 ± 0.02 |
0.04 ± 0.04 |
0.03 ± 0.02 |
0.81 ± 0.05 |
0.58 ± 0.05* |
|
*: significantly different from vehicle control (p < 0.05)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Mode of Action Analysis / Human Relevance Framework
not applicable
Additional information
Justification for classification or non-classification
No classification
The test material did not caus muatagenic effects in bacteria, mammalian cell as well in vivo in mice.
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