Registration Dossier
Registration Dossier
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EC number: 911-739-1 | CAS number: 99402-80-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial Gene Mutation
A weight of evidence assessment was made on the results of the Ames tests conducted wiht PR184. The test substance gave a positive response in strain TA98 with metabolism (pre-incubation, Hamster S9 fraction) in a study conducted at Harlan (2011). Subsequent studies in TA98 conducted did not reproduce this single positive result.
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:
- 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:
- 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:
- 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 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 184 (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 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 184 (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 / 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 184 (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 gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- From 10 NOV 2011 to 16 NOV 2011
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: Prival modification of Ames test but only one strain (TA98) tested.
- Principles of method if other than guideline:
- Bacterial reverse mutation assay
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9 from Phenobarbital/ß-Naphthoflavone induced Wistar rats and hamster liver S9
- Test concentrations with justification for top dose:
- Experiment 1 and 2: 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: 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: 4-Nitro-o-phenylene-diamine
- 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 by rat liver S9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- congo red
- Remarks:
- with metabolic activation by hamster liver S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- experiment 1: plate incorporation with and without rat liver S9 mix
- experiment 2: preincubation with and without hamster liver S9 mix
DURATION
- Preincubation period: 30 min at 30 °C (experiment 2)
- Exposure duration: at least 48 h at 37 °C
DETERMINATION OF CYTOTOXICITY
- Method: other: reductions of spontaneous revertants or cleaning of background lawn - Evaluation criteria:
- The Salmonella typhimurium mutation assay is considered
acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
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 and others) 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:
- not required
- Species / strain:
- S. typhimurium TA 98
- 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 at 333 µg/plate and above in both experiments.
The undissolved particles had no influence on the data recording. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Under the conditions of this study the test item was not mutagenic in TA 98. - Executive summary:
A plate incorporation assay without and with rat liver S9 (experiment I) and the pre-incubation test without and with hamster liver S9 (experiment II) using the Salmonella typhimurium strain TA 98 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:
Experiments 1 and 2: 0, 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 (below the indication factor of 0.5), occurred in the test groups with and without metabolic ativation.
No substantial increase in revertant colony numbers was observed following treatment with the test item at any dose level in experiment 1 in the presence and absence of rat S9 mix.
In experiment 2 an increase in revertant colony numbers was observed in strain TA 98 in the presence of hamster S9 mix. However, the threshold of twice the number of the corresponding solvent control could not be reached and the number of colonies remained within the laboratories historical control range. Therefore, this increase is judged to be biologically irrelevant.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
In conclusion, it can be stated that under the conditions of this study the test item was not mutagenic in TA 98.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From 18 JAN 2007 to 24 JAN 2007
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: Prival modification of Ames test but only one strain (TA98) tested.
- Principles of method if other than guideline:
- Ames test with Prival modification in only one strain (TA 98)
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9 from Phenobarbital/ß-Naphthoflavone induced Wistar rats and hamster liver S9
- Test concentrations with justification for top dose:
- Experiment 1 and 2: 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: 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: 4-Nitro-o-phenylene-diamine
- Remarks:
- TA98 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:
- TA 98 with metabolic activation by rat liver S9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- congo red
- Remarks:
- TA98 with metabolic activation by hamster liver S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- experiment 1: preincubation with and without rat liver S9 mix
- experiment 2: preincubation with hamster liver S9 mix
DURATION
- Preincubation period: 60 min (experiment 1) and 30 min (experiment 2) at 37 °C
- Exposure duration: at least 48 h at 37 °C
DETERMINATION OF CYTOTOXICITY
- Method: other: reductions of spontaneous revertants or cleaning of background lawn - Evaluation criteria:
- The Salmonella typhimurium reverse mutation assay is considered
acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
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) 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:
- not required
- Species / strain:
- S. typhimurium TA 98
- 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 at 33-5000 µg/plate.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Under the conditions of this study the test item was not mutagenic in TA 98. - Executive summary:
A preincubation assay with and without rat liver S9 (experiment I) and with hamster liver S9 (experiment II) using the Salmonella typhimurium strain TA 98 was performed in two independent experiments. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:
Experiments 1 and 2: 0, 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 (below the indication factor of 0.5), occurred in the test groups with and without metabolic ativation. Only in the absence of metabolic activation a minor reduction in the number of revertants was observed.
No substantial increase in revertant colony numbers 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.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- From 10 NOV 2011 to 16 NOV 2011
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: Prival modification of Ames test but only one strain (TA98) tested.
- Principles of method if other than guideline:
- Bacterial reverse mutation assay
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9 from Phenobarbital/ß-Naphthoflavone induced Wistar rats and hamster liver S9
- Test concentrations with justification for top dose:
- Experiment 1 and 2: 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: 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: 4-Nitro-o-phenylene-diamine
- 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 by rat liver S9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- congo red
- Remarks:
- with metabolic activation by hamster liver S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- experiment 1: plate incorporation assay with and without rat liver S9 mix
- experiment 2: preincubation assay with and without hamster liver S9 mix
DURATION
- Preincubation period: 30 min at 30 °C (experiment 2)
- Exposure duration: at least 48 h at 37 °C
DETERMINATION OF CYTOTOXICITY
- Method: other: reductions of spontaneous revertants or cleaning of background lawn - Evaluation criteria:
- The Salmonella typhimurium mutation assay is considered
acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
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 and others) 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:
- not required
- Species / strain:
- S. typhimurium TA 98
- 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 at 1000 µg/plate and above in both experiments.
The undissolved particles had no influence on the data recording. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Under the conditions of this study the test item was not mutagenic in TA 98. - Executive summary:
A plate incorporation assay without and with rat liver S9 (experiment I) and the pre-incubation test without and with hamster liver S9 (experiment II) using the Salmonella typhimurium strain TA 98 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:
Experiments 1 and 2: 0, 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 (below the indication factor of 0.5), occurred in the test groups with and without metabolic ativation.
No substantial increase in revertant colony numbers was observed following treatment with the test item at any dose level in experiment 1 in the presence and absence of rat S9 mix.
In experiment 2 an increase in revertant colony numbers was observed in strain TA 98 in the presence of hamster S9 mix. However, the threshold of twice the number of the corresponding solvent control could not be reached and the number of colonies remained within the laboratories historical control range. Therefore, this increase is judged to be biologically irrelevant.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
In conclusion, it can be stated that under the conditions of this study the test item was not mutagenic in TA 98.
- 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 JUL 2011 to 05 SEP 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD 471 with Prival Modification for Azo Dyes), GLP compliant
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9 from Phenobarbital/ß-Naphthoflavone induced Wistar rats and hamster liver S9
- Test concentrations with justification for top dose:
- Experiment 1 and 2: 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: 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:
- sodium azide
- Remarks:
- TA 1535 and TA100 without metabolic activation
- 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
- Remarks:
- TA1537 and TA98 without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- WP2 uvrA 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:
- all strains with metabolic activation by rat liver S9 and TA 1535, TA1537, TA 100 and WP2 uvrA with metabolic activation by hamster liver S9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- congo red
- Remarks:
- TA98 with metabolic activation by hamster liver S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- experiment 1: plate incorporation with and without rat liver S9 mix
- experiment 2: preincubation with and without hamster liver S9 mix
DURATION
- Preincubation period: 30 min at 30 °C (experiment 2)
- Exposure duration: at least 48 h at 37 °C
DETERMINATION OF CYTOTOXICITY
- Method: other: reductions of spontaneous revertants or cleaning of background lawn - Evaluation criteria:
- The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered
acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
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:
- not required
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Experiment 1
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- other: TA 100, TA 1535, TA 1537, TA 1538, WP2uvrA
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Experiment 2
- 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 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- Experiment 2
- 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 98
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- Experiment 2
- 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 in the overlay agar in the test tubes at 1000-5000 µg/plate in both experiments.
Precipitation on plates at 333-5000 µg/plate in both experiments.
The undissolved particles had no influence on the data recording.
Only minor reductions in the number of revertants (below the indication factor of 0.5) were observed in strain TA 1537 (without S9 mix, exp. 1 and 2) and strain TA 1535 (with S9 mix, exp. 1). - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
positive with metabolic activation in TA 98 with MA in preincubation assay (Prival modification)
negative with metabolic activation in TA 100, TA 1535, TA 1537 and E.coli WP2uvrA in preincubation assay (Prival modification)
negative without metabolic activation in TA 98, TA 100, TA 1535, TA 1537 and E.coli WP2uvrA in preincubation assay (Prival modification)
negative in TA 98, TA 100, TA 1535, TA 1537 and E.coli WP2uvrA with and without MA in plate incorporation assay
The test item was negative in a plate incorporation assay with and without rat liver S9 (experiment I) and a pre-incubation test with and without hamster liver S9 (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2 uvrA, except for a positive result obtained in TA 98 in the pre-incubation assay with hamster liver S9. - Executive summary:
A plate incorporation assay without and with rat liver S9 (experiment I) and the pre-incubation test without and with hamster liver S9 (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2 uvrA 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:
Experiments 1 and 2: 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 (below the indication factor of 0.5), occurred in the test groups with and without metabolic ativation.Only minor reductions in the number of revertants (below the indication factor of 0.5) were observed in strain TA 1537 (without S9 mix, exp. 1 and 2) and strain TA 1535 (with S9 mix, exp. 1).
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 in experiment 1 in the presence and absence of rat S9 mix.
In experiment 2 an increase in revertant colony numbers was observed in strain TA 98 in the presence of hamster S9 mix. The number of colonies reached or exceeded the threshold of twice the number of the corresponding solvent control at 100 µg/plate and above.
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
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
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
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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