Registration Dossier
Registration Dossier
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EC number: 229-107-2 | CAS number: 6410-41-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial Gene Mutation
PR005
During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Chromosome Aberration
PR022
In a guideline study (OECD 473) the test item, suspended in ethanol, was assessed for its potential to induce structural chromosome aberrations in Chinese hamster lung fibroblasts (CHL/IU) with and without metabolic activation.
It was concluded that the test substance is not clastogenic in Chinese hamster lung fibroblast cells (CHL/IU) under the experimental conditions described in the report.
Mammalian Gene Mutation
PR112
The test item was not genotoxic in the mammalian cell gene (HPRT) mutation test in V79 Chinese Hamster cells when tested with and without rat liver S9 metabolic activation at concentrations up to 400 µg/ml.
In vitro micronucleus
PR112
In an in-vitro micronuclus assay in Chinese hamster V79 cells, the test item at concentrations up to 31.3 µg/ml did not induce micronuclei in the absence and presence of metabolic activation. Therefore, Pigment Red 112 can be considered as non-mutagenic in this in vitro test system, when tested up to precipitating concentrations.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: According to Draft Proposal for a new OECD Guideline 487 and GLP
- Justification for type of information:
- See Rationale and Justification for the Analogue Read-Across Approach for the registration of the Nanoform of Pigment Red 5 (Chapter 13)
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Principles of method if other than guideline:
- OECD Guideline Draft Proposal for a new Guideline No. 487, Version 3
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- Micronucleus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks (Greiner,
72632 Frickenhausen, Germany). About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium; Invitrogen GIBCO, 76131 Karlsruhe, Germany) supplemented with 10 % fetal calf serum (FCS; Invitrogen, 76131 Karrlsruhe, Germany). Additionally, the medium was supplemented with 1 % 100x Penicillin/ Streptomycin solution (10.000 Units/mL Penicillin, 10 mg/mL Streptomycin; PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % Amphotericin B (250 µg/mL, PAA Laboratories GmbH, 35091 Cölbe, Germany). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % carbon dioxide (95.5% air). - Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9
- Test concentrations with justification for top dose:
- Exp. I: with and without S9 mix: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL
Exp. II: with S9 mix: 0.3, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3 µg/mL
without S9 mix: 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: regard to the ability to formulate a manageable suspension of the test item in DMSO - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Migrated to IUCLID6: in the absence of S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- Migrated to IUCLID6: in the presence of S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: griseofulvin - in the absence of S9 mix
- Details on test system and experimental conditions:
- Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item.
METHOD OF APPLICATION: in minimal essential medium
DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): May Gruenwald and Giemsa
NUMBER OF REPLICATIONS: 1.5 - 2
NUMBER OF CELLS EVALUATED: 2000
DETERMINATION OF CYTOTOXICITY
- Method: Proliferation Index
OTHER: none
- Evaluation criteria:
- Evaluation of the cultures was performed manually using NIKON microscopes with 40 x oil immersion objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 2000 cells from clones with 2 - 8 cells were scored per test group. The frequency of micronucleated cells was reported as % micronucleated cells.
- Statistics:
Statistical significance can be confirmed by means of the Chi square test.- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The test item, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 24 hours without metabolic activation and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item.
In each experimental group two parallel cultures were set up and 1000 cells per culture were scored for micronuclei.
No influence of the test item on pH value or osmolarity was observed (Exp. I: solvent control 314 mOsm, pH 7.8 versus 354 mOsm and pH 7.8 at 250.0 µg/mL; Exp. II: solvent control 365 mOsm, pH 7.7 versus 369 mOsm and pH 7.7 at 62.5 µg/mL).
In Experiment I test item precipitation in culture medium at the end of treatment was observed at 31.3 µg/mL and above in the absence and presence of metabolic activation. In Experiment II precipitation occurred at 15.6 µg/mL and above in the absence of metabolic activation and at 31.3 µg/mL and above in the presence of metabolic activation.
On the evaluated slides neither in the absence nor in the presence of metabolic activation cytotoxicity measured as reduced proliferation index was observed up to the highest evaluable concentration.
In the absence and presence of metabolic activation no statistically significant or biologically relevant increase in the percentage of micronucleated cells was observed up to the highest evaluable concentrations. The rates of micronucleated cells after treatment with the test item (0.20 - 1.50 %) were close to the range of the solvent control values (0.35 – 1.25 %) and within the range of the laboratory´s historical control data.
Mitomycin C (0.1 µg/mL), Griseofulvin (9.0 µg/mL) and CPA (10.0 and 15.0 µg/mL) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- In an in-vitro micronuclus assay in Chinese hamster V79 cells, the test item at concentrations up to 31.3 µg/ml did not induce micronuclei in the absence and presence of metabolic activation. Therefore, Pigment Red 112 can be considered as non-mutagenic in this in vitro test system, when tested up to precipitating concentrations.
- Executive summary:
The test item, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamsterV79cells in vitro in the absence and presence of metabolic activation by S9 mix. The following study design was performed:
Experiment I
Experiment II
Without S9 mix
With S9 mix
Without S9 mix
With S9 mix
Exposure period
4 hours
4 hours
24 hours
4 hours
Recovery
20 hours
20 hours
0 hours
20 hours
Preparation interval
24 hours
24 hours
24 hours
24 hours
In each experimental group two parallel cultures were analysed and 1000 cells per culture were scored for micronuclei.
The highest applied concentration (250 µg/mL) was chosen with respect to the ability to formulate a homogeneous suspension of the test item in DMSO. The following test item concentrations were applied:
Exp. I: with and without S9 mix: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL
Exp. II: without S9 mix: 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/mL
with S9 mix: 0.3, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3 µg/mL.On the evaluable slides neither in the absence nor in the presence of metabolic activation cytotoxicity measured as reduced proliferation index was observed. Due to strong test item precipitation in culture or on the slides higher concentrations could not be evaluated for cytogenetic damage.
In the presence as well as in the absence of metabolic activation, no biologically relevant increase in the percentage of micronucleated cells was observed after treatment with the test item with respect to the evaluation criteria mentioned in the current guideline forin vitrogenotoxicity studies.
Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in the percentage of micronucleated cells.
In conclusion, it can be stated that under the experimental conditions reported, the test item Pigment Red 112 did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and presence of metabolic activation.
Therefore, Pigment Red 112 is considered to be non-mutagenic in thisin vitrotest system, when tested up to precipitating concentrations.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: According to Draft Proposal for a new OECD Guideline 487 and GLP
- Qualifier:
- according to guideline
- Principles of method if other than guideline:
- OECD Guideline Draft Proposal for a new Guideline No. 487, Version 3
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- Micronucleus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks (Greiner,
72632 Frickenhausen, Germany). About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium; Invitrogen GIBCO, 76131 Karlsruhe, Germany) supplemented with 10 % fetal calf serum (FCS; Invitrogen, 76131 Karrlsruhe, Germany). Additionally, the medium was supplemented with 1 % 100x Penicillin/ Streptomycin solution (10.000 Units/mL Penicillin, 10 mg/mL Streptomycin; PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % Amphotericin B (250 µg/mL, PAA Laboratories GmbH, 35091 Cölbe, Germany). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % carbon dioxide (95.5% air). - Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9
- Test concentrations with justification for top dose:
- Exp. I: with and without S9 mix: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL
Exp. II: with S9 mix: 0.3, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3 µg/mL
without S9 mix: 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: regard to the ability to formulate a manageable suspension of the test item in DMSO - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Migrated to IUCLID6: in the absence of S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- Migrated to IUCLID6: in the presence of S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: griseofulvin - in the absence of S9 mix
- Details on test system and experimental conditions:
- Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item.
METHOD OF APPLICATION: in minimal essential medium
DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): May Gruenwald and Giemsa
NUMBER OF REPLICATIONS: 1.5 - 2
NUMBER OF CELLS EVALUATED: 2000
DETERMINATION OF CYTOTOXICITY
- Method: Proliferation Index
OTHER: none
- Evaluation criteria:
- Evaluation of the cultures was performed manually using NIKON microscopes with 40 x oil immersion objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 2000 cells from clones with 2 - 8 cells were scored per test group. The frequency of micronucleated cells was reported as % micronucleated cells.
- Statistics:
Statistical significance can be confirmed by means of the Chi square test.- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The test item, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 24 hours without metabolic activation and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item.
In each experimental group two parallel cultures were set up and 1000 cells per culture were scored for micronuclei.
No influence of the test item on pH value or osmolarity was observed (Exp. I: solvent control 314 mOsm, pH 7.8 versus 354 mOsm and pH 7.8 at 250.0 µg/mL; Exp. II: solvent control 365 mOsm, pH 7.7 versus 369 mOsm and pH 7.7 at 62.5 µg/mL).
In Experiment I test item precipitation in culture medium at the end of treatment was observed at 31.3 µg/mL and above in the absence and presence of metabolic activation. In Experiment II precipitation occurred at 15.6 µg/mL and above in the absence of metabolic activation and at 31.3 µg/mL and above in the presence of metabolic activation.
On the evaluated slides neither in the absence nor in the presence of metabolic activation cytotoxicity measured as reduced proliferation index was observed up to the highest evaluable concentration.
In the absence and presence of metabolic activation no statistically significant or biologically relevant increase in the percentage of micronucleated cells was observed up to the highest evaluable concentrations. The rates of micronucleated cells after treatment with the test item (0.20 - 1.50 %) were close to the range of the solvent control values (0.35 – 1.25 %) and within the range of the laboratory´s historical control data.
Mitomycin C (0.1 µg/mL), Griseofulvin (9.0 µg/mL) and CPA (10.0 and 15.0 µg/mL) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- In an in-vitro micronuclus assay in Chinese hamster V79 cells, the test item at concentrations up to 31.3 µg/ml did not induce micronuclei in the absence and presence of metabolic activation. Therefore, Pigment Red 112 can be considered as non-mutagenic in this in vitro test system, when tested up to precipitating concentrations.
- Executive summary:
The test item, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamsterV79cells in vitro in the absence and presence of metabolic activation by S9 mix. The following study design was performed:
Experiment I
Experiment II
Without S9 mix
With S9 mix
Without S9 mix
With S9 mix
Exposure period
4 hours
4 hours
24 hours
4 hours
Recovery
20 hours
20 hours
0 hours
20 hours
Preparation interval
24 hours
24 hours
24 hours
24 hours
In each experimental group two parallel cultures were analysed and 1000 cells per culture were scored for micronuclei.
The highest applied concentration (250 µg/mL) was chosen with respect to the ability to formulate a homogeneous suspension of the test item in DMSO. The following test item concentrations were applied:
Exp. I: with and without S9 mix: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL
Exp. II: without S9 mix: 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/mL
with S9 mix: 0.3, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3 µg/mL.On the evaluable slides neither in the absence nor in the presence of metabolic activation cytotoxicity measured as reduced proliferation index was observed. Due to strong test item precipitation in culture or on the slides higher concentrations could not be evaluated for cytogenetic damage.
In the presence as well as in the absence of metabolic activation, no biologically relevant increase in the percentage of micronucleated cells was observed after treatment with the test item with respect to the evaluation criteria mentioned in the current guideline forin vitrogenotoxicity studies.
Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in the percentage of micronucleated cells.
In conclusion, it can be stated that under the experimental conditions reported, the test item Pigment Red 112 did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and presence of metabolic activation.
Therefore, Pigment Red 112 is considered to be non-mutagenic in thisin vitrotest system, when tested up to precipitating concentrations.
- 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
- Study period:
- 29 January - 14 March 2008
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study performed according to OECD test guideline and GLP
- Justification for type of information:
- See Rationale and Justification for the Analogue Read-Across Approach for the registration of the Nanoform of Pigment Red 5 (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)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: minimum essential medium containing 10% fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix (phenobarbital/beta-naphthoflavone-induced rat liver protein with cofactors)
- Test concentrations with justification for top dose:
- 3.1, 6.3, 12.5, 25.0, 50.0 and 400 µg/ml with and without metabolic activation
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Migrated to IUCLID6: w/o metabolic activation; 7,12-Dimethylbenz(a)anthracene with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium (4h-incubation without serum, 24 h incubations with serum)
DURATION
- Preincubation period: none
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells):
SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution
NUMBER OF REPLICATIONS: 5/experiment, 2 experiments
NUMBER OF CELLS EVALUATED: colonies >50 cells
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- test item classified as positive for mutagenicity if induces either concentration-related increase of mutant frequency or reproducible and positive response at one of the test points (at least three times above the spontaneous mutation frequency)
- Statistics:
- linear regerssion on dose-dependeny of mutant frequencies using SYSTAT statistics software
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- precipitation was observed at 12.5 µg/ml and higher (Exp. I without S9-mix), at 25.0 µg/ml and higher (Exp. I with S9-mix and Exp. II)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: negligible decrease (0.1 pH-units) at highest dose
- Effects of osmolality: negligible increase (+3 mOsm) at highest dose - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative with and without metabolic activation
The test item was not genotoxic in the mammalian cell gene (HPRT) mutation test in V79 Chinese Hamster cells when tested with and without rat liver S9 metabolic activation at concentrations up to 400 µg/ml. - 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:
- 29 January - 14 March 2008
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study performed according to OECD test guideline and GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: minimum essential medium containing 10% fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix (phenobarbital/beta-naphthoflavone-induced rat liver protein with cofactors)
- Test concentrations with justification for top dose:
- 3.1, 6.3, 12.5, 25.0, 50.0 and 400 µg/ml with and without metabolic activation
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Migrated to IUCLID6: w/o metabolic activation; 7,12-Dimethylbenz(a)anthracene with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium (4h-incubation without serum, 24 h incubations with serum)
DURATION
- Preincubation period: none
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells):
SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution
NUMBER OF REPLICATIONS: 5/experiment, 2 experiments
NUMBER OF CELLS EVALUATED: colonies >50 cells
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- test item classified as positive for mutagenicity if induces either concentration-related increase of mutant frequency or reproducible and positive response at one of the test points (at least three times above the spontaneous mutation frequency)
- Statistics:
- linear regerssion on dose-dependeny of mutant frequencies using SYSTAT statistics software
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- precipitation was observed at 12.5 µg/ml and higher (Exp. I without S9-mix), at 25.0 µg/ml and higher (Exp. I with S9-mix and Exp. II)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: negligible decrease (0.1 pH-units) at highest dose
- Effects of osmolality: negligible increase (+3 mOsm) at highest dose - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative with and without metabolic activation
The test item was not genotoxic in the mammalian cell gene (HPRT) mutation test in V79 Chinese Hamster cells when tested with and without rat liver S9 metabolic activation at concentrations up to 400 µg/ml. - Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD 473) and according to GLP.
- Justification for type of information:
- See Rationale and Justification for the Analogue Read-Across Approach for the registration of the Nanoform of Pigment Red 5 (Chapter 13)
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- other: Chinese hamster lung cells (CHL/IU)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data - Metabolic activation:
- with and without
- Metabolic activation system:
- liver S9 mix from phenobarbital and 5,6-benzoflavone induced rats
- Test concentrations with justification for top dose:
- cell growth inhibition test: 0, 8.4, 16.8, 33.6, 67.2, 134.4, 268.8, 537.5, 1075, 2150, 4300 µg/mL
chromosomal abberation test: 0, 37.5, 75, 150, 300, 600 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test substance is insolube in water, DMSO and acetone - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- for continuous treatment and short treatment in absence of metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: dimethylnitrosamine
- Remarks:
- for short treatment method in the presence of metabolic activation
- Details on test system and experimental conditions:
- DURATION
- Exposure duration:
continuous treatment: 24 or 48 h
short treatment: 6 h
- Expression time (cells in growth medium):
short treatment: 18 h
- Fixation time (start of exposure up to fixation or harvest of cells):
continuous treatment: 24 or 48 h
short treatment: 24h
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (2 h before completion of incubation)
STAIN (for cytogenetic assays): Giemsa solution
NUMBER OF REPLICATIONS: Doubling time: 16.2 h
NUMBER OF CELLS EVALUATED: 200 cells per concentration
DETERMINATION OF CYTOTOXICITY
- Method: cell survival ratio
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
OTHER EXAMINATIONS:
- Determination of polyploidy: yes - Evaluation criteria:
- The final judgement on the outcome of the test was made as follows: when the incidence of cells with numerical or structural aberrations induced by the test article was lower than 5, the result was assessed as negative; from 5-10% (exclusive of 10%), equivocal; and 10% or higher with a concentration-dependent rise, positive. The incidence of cells having structural abberations with gaps and of cells without gaps were calculated separately, and cells without gaps were assessed for chromosomal aberrarions.
- Statistics:
- No significance tests were performed, since the incidence of cells with chromosomal aberrations was assessed according to the evaluation criteria described above.
- Species / strain:
- other: Chinese hamster lung cells (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- precipitation occurred at concentrations of 268.8 µg/mL and higher
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation occurred at concentrations of 268.8 µg/mL and higher
ADDITIONAL INFORMATION ON CYTOTOXICITY:
in the cell growth inhibition test no cytotoxicity was noted. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test item is not clastogenic in Chinese hamster lung fibroblast (CHL/IU) cells under the experimental conditions described in the study. - Executive summary:
In a guideline study (OECD 473) the test item, suspended in ethanol, was assessed for its potential to induce structural chromosome aberrations in Chinese hamster lung fibroblasts (CHL/IU) with and without metabolic activation.
Prior to the chromosomal aberration test, a cell growth inhibition test was performed to determine test concentrations for the chromosomal aberration test. No cytotoxicity was noted at 4300 µg/mL, the highest concentration tested. Precipitations were noted at concentrations of 268.8 µg/mL and higher.
In the chromosomal aberration test, concentrations of 0, 37.5, 75, 150, 300 and 600 µg/mL were tested. A continuous exposure (24 or 48 h) and a short time exposure (6 h with 18 h expression time) in the presence and absence of S9-mix were performed.
At least 200 metaphases per culture were evaluated for structural chromosome aberrations.
Appropriate mutagens were used as positive controls. They produced markedly positive results.
It was concluded that the test substance is not clastogenic in Chinese hamster lung fibroblast cells (CHL/IU) under the experimental conditions described in the report.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD 473) and according to GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- other: Chinese hamster lung cells (CHL/IU)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data - Metabolic activation:
- with and without
- Metabolic activation system:
- liver S9 mix from phenobarbital and 5,6-benzoflavone induced rats
- Test concentrations with justification for top dose:
- cell growth inhibition test: 0, 8.4, 16.8, 33.6, 67.2, 134.4, 268.8, 537.5, 1075, 2150, 4300 µg/mL
chromosomal abberation test: 0, 37.5, 75, 150, 300, 600 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test substance is insolube in water, DMSO and acetone - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- for continuous treatment and short treatment in absence of metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: dimethylnitrosamine
- Remarks:
- for short treatment method in the presence of metabolic activation
- Details on test system and experimental conditions:
- DURATION
- Exposure duration:
continuous treatment: 24 or 48 h
short treatment: 6 h
- Expression time (cells in growth medium):
short treatment: 18 h
- Fixation time (start of exposure up to fixation or harvest of cells):
continuous treatment: 24 or 48 h
short treatment: 24h
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (2 h before completion of incubation)
STAIN (for cytogenetic assays): Giemsa solution
NUMBER OF REPLICATIONS: Doubling time: 16.2 h
NUMBER OF CELLS EVALUATED: 200 cells per concentration
DETERMINATION OF CYTOTOXICITY
- Method: cell survival ratio
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
OTHER EXAMINATIONS:
- Determination of polyploidy: yes - Evaluation criteria:
- The final judgement on the outcome of the test was made as follows: when the incidence of cells with numerical or structural aberrations induced by the test article was lower than 5, the result was assessed as negative; from 5-10% (exclusive of 10%), equivocal; and 10% or higher with a concentration-dependent rise, positive. The incidence of cells having structural abberations with gaps and of cells without gaps were calculated separately, and cells without gaps were assessed for chromosomal aberrarions.
- Statistics:
- No significance tests were performed, since the incidence of cells with chromosomal aberrations was assessed according to the evaluation criteria described above.
- Species / strain:
- other: Chinese hamster lung cells (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- precipitation occurred at concentrations of 268.8 µg/mL and higher
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation occurred at concentrations of 268.8 µg/mL and higher
ADDITIONAL INFORMATION ON CYTOTOXICITY:
in the cell growth inhibition test no cytotoxicity was noted. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test item is not clastogenic in Chinese hamster lung fibroblast (CHL/IU) cells under the experimental conditions described in the study. - Executive summary:
In a guideline study (OECD 473) the test item, suspended in ethanol, was assessed for its potential to induce structural chromosome aberrations in Chinese hamster lung fibroblasts (CHL/IU) with and without metabolic activation.
Prior to the chromosomal aberration test, a cell growth inhibition test was performed to determine test concentrations for the chromosomal aberration test. No cytotoxicity was noted at 4300 µg/mL, the highest concentration tested. Precipitations were noted at concentrations of 268.8 µg/mL and higher.
In the chromosomal aberration test, concentrations of 0, 37.5, 75, 150, 300 and 600 µg/mL were tested. A continuous exposure (24 or 48 h) and a short time exposure (6 h with 18 h expression time) in the presence and absence of S9-mix were performed.
At least 200 metaphases per culture were evaluated for structural chromosome aberrations.
Appropriate mutagens were used as positive controls. They produced markedly positive results.
It was concluded that the test substance is not clastogenic in Chinese hamster lung fibroblast cells (CHL/IU) under the experimental conditions described in the report.
- 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 30 OCT 2012 to 19 NOV 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD TG 471) with Prival modification for azo-dyes, according to GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/ß-Naphthoflavone induced rat liver S9 (Experiment I), non induced hamster liver S9 (Experiment II)
- Test concentrations with justification for top dose:
- Experiment I: 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: suspended in DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide, NaN3 used with Strains: TA 1535 and TA 100
- Remarks:
- Without metabolic activation (Experiment I and II)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylene-diamine, 4-NOPD used with Strains: TA 1537, TA 98
- Remarks:
- Without metabolic activation (Experiment I and II)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: methylmethanesulfonate, MMS used with Strain: WP2 uvrA
- Remarks:
- Without metabolic activation (Experiment I and II)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene, 2-AA used with Strains:TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
- Remarks:
- With metabolic activation (rat liver S9, Experiment I)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: congo red, used with strain TA 98 @ 500 µg/plate; 2-aminoanthracene used with strain TA1535, TA1537, TA100 and with strain WP2uvrA
- Remarks:
- with metabolic activation (hamster liver S9, Experiment II)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar, plate incorporation (Experiment I), preincubation (Experiment II)
DURATION
- Preincubation period: 30 minutes
- Exposure duration: at least 48 hours
NUMBER OF REPLICATIONS: 3 plates
DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.
The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major draw¬back an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
8.4.1 Rat Liver S9 (Preparation by Harlan CCR)
Phenobarbital/ß-naphthoflavone induced rat liver S9 will be used as the metabolic activation system. The S9 is prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin; 22335 Hamburg, Germany) and by peroral administrations of ß-naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.
The protein concentration in the S9 preparation was 31.5 mg/mL (lot no. R 121012).
8.4.2 Hamster Liver S9 (Preparation by Harlan CCR)
The S9 liver microsomal fraction was prepared from the liver of 7 - 8 weeks old male Syrian golden hamsters.
After decapitation of the anaesthetised animals the livers of the animals was removed, washed in 0.1 M sodium phosphate buffer pH 7.4, 0.25 M sucrose and 1 mM disodium EDTA in deionised water and homogenised. The homogenate, diluted 1+3 in sodium phosphate buffer was centrifuged at 9,000 g for 25 minutes at 4 °C. Aliquots of the supernatant were frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as congo red.
The protein concentration in the S9 preparation was 23.5 mg/mL (lot no. H 020712).
8.4.3 Rat S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.
8.4.4 Hamster S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 30% v/v. The concentrated cofactor solution yields the following concentrations in the S9 mix:
8.0 mM MgCl2
33.0 mM KCl
20.0 mM Glucose-6-phosphate
2.8 units/ml Glucose-6-phosphate-dehydrogenase
4.0 mM NADP
2.0 mM NADH
2.0 mM FMN
in 100 mM Sodium-Ortho-Phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. and Prival and Mitchell .
- Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
- Species / strain:
- other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in both experiments. Precipitation of the test item on the incubated agar plates was observed from 333 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabol¬ic activation. - Remarks on result:
- other: other: reverse mutation assay
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay. - Executive summary:
This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I), and the pre-incubation test with hamster liver S9 (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.
The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations in both experiments:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
Referenceopen allclose all
Summary of results of the micronucleus test with test item
Exp. |
Preparation |
Test item |
Proliferation |
Micronucleated |
interval |
concentration |
Index |
Cells* |
|
in µg/mL |
in % |
|||
Exposure period 4 hrs without S9 mix |
||||
I |
24 hrs |
Solvent control1 |
2.92 |
0.35 |
Positive control2 |
2.60 |
6.90S |
||
7.8 |
2.95 |
0.35 |
||
15.6 |
3.02 |
0.45 |
||
31.3P |
2.95 |
0.65 |
||
Exposure period 24 hrs without S9 mix |
||||
II |
24 hrs |
Solvent control1 |
3.04 |
1.25 |
Positive control2 |
2.58 |
11.05S |
||
Positive control3 |
2.55 |
24.20S |
||
3.9 |
3.08 |
0.70 |
||
7.8 |
2.99 |
1.15 |
||
15.6P |
2.97 |
1.50 |
||
Exposure period 4 hrs with S9 mix |
||||
I |
24 hrs |
Solvent control1 |
2.53 |
0.75 |
Positive control4 |
1.86 |
4.95S |
||
2.0 |
2.61 |
0.55 |
||
3.9 |
2.69 |
0.70 |
||
7.8 |
2.67 |
0.20 |
||
II |
24 hrs |
Solvent control1 |
2.09 |
0.95 |
Positive control5 |
1.66 |
7.25S |
||
2.0 |
2.04 |
0.50 |
||
3.9 |
2.11 |
0.40 |
||
7.8 |
2.00 |
0.25 |
||
* The number of micronucleated cells was determined of each test group in a sample of 2000 cells
P Precipitation occurred at the end of treatment
S Number of micronucleated cells statistically significantly higher than corresponding control values
1 DMSO 0.5% (v/v)
2 Mitomycin C 0.1 µg/mL
3 Griseofulvin 9.0 µg/mL
4 CPA 10.0 µg/mL
5 CPA 15.0 µg/mL
Summary of results of the micronucleus test with test item
Exp. |
Preparation |
Test item |
Proliferation |
Micronucleated |
interval |
concentration |
Index |
Cells* |
|
in µg/mL |
in % |
|||
Exposure period 4 hrs without S9 mix |
||||
I |
24 hrs |
Solvent control1 |
2.92 |
0.35 |
Positive control2 |
2.60 |
6.90S |
||
7.8 |
2.95 |
0.35 |
||
15.6 |
3.02 |
0.45 |
||
31.3P |
2.95 |
0.65 |
||
Exposure period 24 hrs without S9 mix |
||||
II |
24 hrs |
Solvent control1 |
3.04 |
1.25 |
Positive control2 |
2.58 |
11.05S |
||
Positive control3 |
2.55 |
24.20S |
||
3.9 |
3.08 |
0.70 |
||
7.8 |
2.99 |
1.15 |
||
15.6P |
2.97 |
1.50 |
||
Exposure period 4 hrs with S9 mix |
||||
I |
24 hrs |
Solvent control1 |
2.53 |
0.75 |
Positive control4 |
1.86 |
4.95S |
||
2.0 |
2.61 |
0.55 |
||
3.9 |
2.69 |
0.70 |
||
7.8 |
2.67 |
0.20 |
||
II |
24 hrs |
Solvent control1 |
2.09 |
0.95 |
Positive control5 |
1.66 |
7.25S |
||
2.0 |
2.04 |
0.50 |
||
3.9 |
2.11 |
0.40 |
||
7.8 |
2.00 |
0.25 |
||
* The number of micronucleated cells was determined of each test group in a sample of 2000 cells
P Precipitation occurred at the end of treatment
S Number of micronucleated cells statistically significantly higher than corresponding control values
1 DMSO 0.5% (v/v)
2 Mitomycin C 0.1 µg/mL
3 Griseofulvin 9.0 µg/mL
4 CPA 10.0 µg/mL
5 CPA 15.0 µg/mL
Results of V79/HPRT assay with test item
Concentration [microgram/mL] |
mutant colonies per 10^-6 cells** |
mutant colonies per 10^6 cells** |
Cytotoxicity |
Remarks |
|
— MA |
+ MA |
|
|
0* |
7.6, 6.2 |
5.2, 21.2 |
no |
Exp. I |
3.1 |
13.6, 8.5 |
11.7, 13.4 |
no |
|
6.3 |
7.8, 14.7 |
8.6, 26.8 |
no |
|
12.5 |
12.3, 8.6 |
13.4, 19.5 |
no |
|
25.0 |
12.3, 10.9 |
7.4, 22.3 |
no |
|
400 |
15.9, 10.6 |
13.1, 12.5 |
no |
|
Positive control*** |
151.7, 87.5 |
1239.9, 1528.8 |
-MA: no; +MA: yes |
|
0* |
13.9, 11.3 |
- |
no |
Exp. II |
3.1 |
11.4, 6.1 |
- |
no |
|
6.3 |
12.2, 7.7 |
- |
no |
|
12.5 |
17.1, 12.9 |
- |
no |
|
25.0 |
11.0, 9.6 |
- |
no |
|
400 |
5.5, 7.7 |
- |
no |
|
Positive control*** |
186.6,. 136.9 |
- |
yes |
|
*solvent control with DMSO
** mean of 5 plates each in culture I and culture II
***without MA: EMS, with MA: DMBA
Results of V79/HPRT assay with test item
Concentration [microgram/mL] |
mutant colonies per 10^-6 cells** |
mutant colonies per 10^6 cells** |
Cytotoxicity |
Remarks |
|
— MA |
+ MA |
|
|
0* |
7.6, 6.2 |
5.2, 21.2 |
no |
Exp. I |
3.1 |
13.6, 8.5 |
11.7, 13.4 |
no |
|
6.3 |
7.8, 14.7 |
8.6, 26.8 |
no |
|
12.5 |
12.3, 8.6 |
13.4, 19.5 |
no |
|
25.0 |
12.3, 10.9 |
7.4, 22.3 |
no |
|
400 |
15.9, 10.6 |
13.1, 12.5 |
no |
|
Positive control*** |
151.7, 87.5 |
1239.9, 1528.8 |
-MA: no; +MA: yes |
|
0* |
13.9, 11.3 |
- |
no |
Exp. II |
3.1 |
11.4, 6.1 |
- |
no |
|
6.3 |
12.2, 7.7 |
- |
no |
|
12.5 |
17.1, 12.9 |
- |
no |
|
25.0 |
11.0, 9.6 |
- |
no |
|
400 |
5.5, 7.7 |
- |
no |
|
Positive control*** |
186.6,. 136.9 |
- |
yes |
|
*solvent control with DMSO
** mean of 5 plates each in culture I and culture II
***without MA: EMS, with MA: DMBA
Experiment I
Study Name: 1513300 |
Study Code: Harlan CCR 1513300 |
Experiment: 1513300 VV Plate |
Date Plated: 30/10/2012 |
Assay Conditions: |
Date Counted: 02/11/2012 |
Metabolic Activation |
Test Group |
Dose Level (per plate) |
|
Revertant Colony Counts (Mean ±SD) |
||||
|
|
|
|
|
|
|
|
|
|
|
|
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|
|
|
|
|
|
|
|
|
Without Activation |
DMSO |
|
|
12 ± 2B M |
9 ± 1 |
31 ± 3 |
97 ± 4 |
49 ± 9 |
Untreated |
|
|
14 ± 3B M |
18 ± 7 |
28 ± 1 |
109 ± 10 |
59 ± 10 |
|
Test item |
3 µg |
|
12 ± 4B M |
8 ± 1 |
28 ± 4 |
100 ± 15 |
47 ± 5 |
|
|
10 µg |
|
9 ± 1B M |
11 ± 2 |
29 ± 6 |
104 ± 6 |
47 ± 4 |
|
|
33 µg |
|
11 ± 2B M |
10 ± 1 |
26 ± 4 |
116 ± 18 |
51 ± 12 |
|
|
100 µg |
|
10 ± 4B M |
14 ± 1 |
28 ± 5 |
122 ± 3 |
49 ± 0 |
|
|
333 µg |
|
10 ± 2B M P |
12 ± 4P |
29 ± 5P |
106 ± 25P |
42 ± 6P |
|
|
1000 µg |
|
11 ± 3B M P |
13 ± 5P |
30 ± 3P |
93 ± 10P |
43 ± 1P |
|
|
2500 µg |
|
8 ± 3B M P |
8 ± 2P M |
26 ± 2P M |
102 ± 4P M |
46 ± 4P M |
|
|
5000 µg |
|
8 ± 2M B P |
7 ± 3P M |
17 ± 3P M |
96 ± 5P M |
34 ± 2P M |
|
NaN3 |
10 µg |
|
1045 ± 61B M |
|
|
2156 ± 131 |
|
|
4-NOPD |
10 µg |
|
|
|
335 ± 19 |
|
|
|
4-NOPD |
50 µg |
|
|
67 ± 6 |
|
|
|
|
MMS |
3.0 µL |
|
|
|
|
|
880 ± 18 |
|
|
|
|
|
|
|
|
|
|
With Activation |
DMSO |
|
|
16 ± 4B M |
22 ± 2 |
37 ± 6 |
127 ± 2 |
51 ± 10 |
Untreated |
|
|
22 ± 4B M |
26 ± 3 |
49 ± 6 |
132 ± 8 |
59 ± 4 |
|
Test item |
3 µg |
|
14 ± 1B M |
21 ± 7 |
39 ± 7 |
105 ± 5 |
51 ± 9 |
|
|
10 µg |
|
17 ± 3B M |
23 ± 1 |
42 ± 6 |
108 ± 6 |
55 ± 2 |
|
|
33 µg |
|
12 ± 4B M |
17 ± 3 |
37 ± 9 |
115 ± 4 |
55 ± 12 |
|
|
100 µg |
|
14 ± 4B M |
23 ± 6 |
35 ± 7 |
123 ± 17 |
49 ± 13 |
|
|
333 µg |
|
19 ± 4B M P |
17 ± 3P |
40 ± 3P |
107 ± 11P |
55 ± 11P |
|
|
1000 µg |
|
14 ± 2B M P |
20 ± 2P |
40 ± 6P |
104 ± 9P |
57 ± 15P |
|
|
2500 µg |
|
13 ± 2B M P |
14 ± 3P M |
27 ± 3P M |
97 ± 13P M |
48 ± 4P M |
|
|
5000 µg |
|
12 ± 2M B P |
14 ± 1P M |
21 ± 2P M |
72 ± 4P M |
33 ± 6P M |
|
2-AA |
2.5 µg |
|
443 ± 41B M |
162 ± 3 |
1448 ± 84 |
1323 ± 75 |
|
|
2-AA |
10.0 µg |
|
|
|
|
|
189 ± 2 |
|
|
|
|
|
|
|
|
|
|
Key to Positive Controls |
Key to Plate Postfix Codes |
||
|
|
||
NaN3 2-AA 4-NOPD MMS |
sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate |
P M B |
Precipitate Manual count Extensive bacterial growth |
Experiment II
Study Name: 1513300 |
Study Code: Harlan CCR 1513300 |
Experiment: 1513300 HV2 Pre |
Date Plated: 13/11/2012 |
Assay Conditions: |
Date Counted: 19/11/2012 |
Metabolic Activation |
Test Group |
Dose Level (per plate) |
|
Revertant Colony Counts (Mean ±SD) |
||||
|
|
|
|
|
|
|
|
|
|
|
|
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|
|
|
|
|
|
|
|
|
Without Activation |
DMSO |
|
|
14 ± 3 |
21 ± 5 |
23 ± 2 |
104 ± 11 |
47 ± 8 |
Untreated |
|
|
19 ± 5 |
23 ± 5 |
24 ± 6 |
114 ± 1 |
43 ± 2 |
|
Test item |
3 µg |
|
14 ± 5 |
23 ± 6 |
24 ± 4 |
85 ± 5 |
46 ± 4 |
|
|
10 µg |
|
12 ± 6 |
17 ± 2 |
17 ± 2 |
99 ± 10 |
44 ± 4 |
|
|
33 µg |
|
13 ± 4 |
24 ± 4 |
24 ± 6 |
95 ± 3 |
49 ± 0 |
|
|
100 µg |
|
13 ± 2 |
21 ± 1 |
26 ± 6 |
103 ± 6 |
54 ± 2 |
|
|
333 µg |
|
8 ± 1P |
26 ± 5P |
23 ± 4P |
94 ± 12P |
41 ± 9P |
|
|
1000 µg |
|
13 ± 4P |
21 ± 0P |
22 ± 4P |
84 ± 6P |
53 ± 7P |
|
|
2500 µg |
|
9 ± 5P |
26 ± 6P M |
17 ± 5P M |
88 ± 10P M |
42 ± 5P M |
|
|
5000 µg |
|
10 ± 2P M |
25 ± 4P M |
17 ± 2P M |
100 ± 12P M |
39 ± 4P M |
|
NaN3 |
10 µg |
|
1985 ± 51 |
|
|
2245 ± 30 |
|
|
4-NOPD |
10 µg |
|
|
|
363 ± 17 |
|
|
|
4-NOPD |
50 µg |
|
|
79 ± 2 |
|
|
|
|
MMS |
3 µL |
|
|
|
|
|
855 ± 62 |
|
|
|
|
|
|
|
|
|
|
With Activation |
DMSO |
|
|
22 ± 5 |
29 ± 1 |
44 ± 4 |
115 ± 6 |
40 ± 4 |
Untreated |
|
|
16 ± 7 |
28 ± 0 |
49 ± 2 |
133 ± 12 |
38 ± 2 |
|
Test item |
3 µg |
|
20 ± 6 |
35 ± 7 |
46 ± 3 |
107 ± 18 |
38 ± 5 |
|
|
10 µg |
|
19 ± 6 |
32 ± 5 |
47 ± 6 |
109 ± 15 |
41 ± 6 |
|
|
33 µg |
|
16 ± 2 |
33 ± 6 |
47 ± 6 |
102 ± 7 |
31 ± 4 |
|
|
100 µg |
|
21 ± 2 |
30 ± 3 |
46 ± 7 |
125 ± 10 |
36 ± 9 |
|
|
333 µg |
|
17 ± 5P |
34 ± 7P |
52 ± 5P |
104 ± 11P |
40 ± 5P |
|
|
1000 µg |
|
14 ± 4P |
25 ± 7P |
33 ± 4P |
98 ± 7P |
37 ± 7P |
|
|
2500 µg |
|
21 ± 3P M |
24 ± 5P M |
35 ± 6P M |
112 ± 9P M |
29 ± 3P M |
|
|
5000 µg |
|
15 ± 3P M |
26 ± 5P M |
35 ± 2P M |
106 ± 11P M |
30 ± 7P M |
|
2-AA |
2.5 µg |
|
|
|
|
3398 ± 54 |
|
|
2-AA |
2.5 µg |
|
414 ± 45 |
495 ± 32 |
|
|
|
|
2-AA |
10 µg |
|
|
|
|
|
669 ± 91 |
|
Congo red |
500 µg |
|
|
|
587 ± 52 |
|
|
|
|
|
|
|
|
|
|
|
|
Key to Positive Controls |
Key to Plate Postfix Codes |
||
|
|
||
NaN3 2-AA 4-NOPD Congo red MMS |
sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine Congo red methyl methane sulfonate |
P M |
Precipitate Manual count |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
No Classification, as no adverse effects observed.
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