Registration Dossier
Registration Dossier
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EC number: 250-830-4 | CAS number: 31837-42-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test item did not cause mutagenic or clastogenic effects in vitro in bacteria as well as mammalian cells
Link to relevant study records
- 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 8 SEP 2004 to 27 SEP 2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD TG 471)
- 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 (induced with phenobarbital/beta-naphtoflavone; experiment I); hamster liver S9 (non-induced; experiment II)
- Test concentrations with justification for top dose:
- Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent 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 (TA 1535 and TA 100), 4-Nitro-o-phenylene-diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA)
- 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 (for all strains)
- Remarks:
- with metabolic activation (rat liver S9)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (TA 1535, TA 100, TA 1537, WP2 uvrA), congo red (TA 98)
- Remarks:
- with metabolic activation (hamster liver S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Experiment I: plate incorporation assay without and with induced rat liver S9 mix (induction with phenobarbital/beta-naphthoflavone)
Experiment II: preincubation assay without and with non-induced hamster liver S9 mix
DURATION
- Preincubation period: Experiment II: 30° C for 30 minutes
- Exposure duration: at least 48 hours at 37° C
NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including the control - 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, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent colony 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 thresshold is regarded as an indication of mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative annd solvent controls such an increase is not considered biologically relevant. - Statistics:
- Arithmetic means and standard deviation of the counted colonies were calculated.
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In experiment II, slight toxic effects were observed in strain TA 1535 (2500 µg/plate), TA 1537 (1000, 2500 µg/plate), and TA 98 (2500 µg/plate) in the absence of metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- COMPARISON WITH HISTORICAL CONTROL DATA:
The number of colonies were slightly above the historical control range in strains TA 1535 (solvent control experiment I), TA 1537 (negative control exp. I and II, solvent control exp. II), TA 98 (negative and solvent control exp. II) with metabolic activation, and WP2 uvrA (negative control experiment I) without metabolic activation. These deviations are considered to be based upon biologically irrelevant fluctuations in the number of colonies and have no detrimental impact on the outcome of the study.
The historical range of positive controls was exceeded in strain TA 1535 (Exp. II) with metabolic activation. This effect indicates the sensitivity of the strain rather than compromising the assay. In strains TA 1537, TA 100 and WP2 uvrA with metabolic activation in strain TA 1535 without metabolic activation of the second experiment the historical range of positive controls was just not reached. This minor effects were judged to represent fluctuations. The threshold of two times (TA 100, WP2 uvrA) and three times (TA 1535, TA 1537) of the corresponding solvent control was exceeded by far so the test was considered valid. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results:
negative
The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation. - Executive summary:
Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation assay. Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay without and with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the concentrations 33, 100, 333, 1000, 2500 and 5000 µg/plate.
The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- From 8 SEP 2004 to 27 SEP 2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD TG 471)
- Justification for type of information:
- See read across justification document in chapter 13
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 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 (induced with phenobarbital/beta-naphtoflavone; experiment I); hamster liver S9 (non-induced; experiment II)
- Test concentrations with justification for top dose:
- Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent 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 (TA 1535 and TA 100), 4-Nitro-o-phenylene-diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA)
- 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 (for all strains)
- Remarks:
- with metabolic activation (rat liver S9)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (TA 1535, TA 100, TA 1537, WP2 uvrA), congo red (TA 98)
- Remarks:
- with metabolic activation (hamster liver S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Experiment I: plate incorporation assay without and with induced rat liver S9 mix (induction with phenobarbital/beta-naphthoflavone)
Experiment II: preincubation assay without and with non-induced hamster liver S9 mix
DURATION
- Preincubation period: Experiment II: 30° C for 30 minutes
- Exposure duration: at least 48 hours at 37° C
NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including the control - 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, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent colony 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 thresshold is regarded as an indication of mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative annd solvent controls such an increase is not considered biologically relevant. - Statistics:
- Arithmetic means and standard deviation of the counted colonies were calculated.
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In experiment II, slight toxic effects were observed in strain TA 1535 (2500 µg/plate), TA 1537 (1000, 2500 µg/plate), and TA 98 (2500 µg/plate) in the absence of metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- COMPARISON WITH HISTORICAL CONTROL DATA:
The number of colonies were slightly above the historical control range in strains TA 1535 (solvent control experiment I), TA 1537 (negative control exp. I and II, solvent control exp. II), TA 98 (negative and solvent control exp. II) with metabolic activation, and WP2 uvrA (negative control experiment I) without metabolic activation. These deviations are considered to be based upon biologically irrelevant fluctuations in the number of colonies and have no detrimental impact on the outcome of the study.
The historical range of positive controls was exceeded in strain TA 1535 (Exp. II) with metabolic activation. This effect indicates the sensitivity of the strain rather than compromising the assay. In strains TA 1537, TA 100 and WP2 uvrA with metabolic activation in strain TA 1535 without metabolic activation of the second experiment the historical range of positive controls was just not reached. This minor effects were judged to represent fluctuations. The threshold of two times (TA 100, WP2 uvrA) and three times (TA 1535, TA 1537) of the corresponding solvent control was exceeded by far so the test was considered valid. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results:
negative
The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation. - Executive summary:
Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation assay. Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay without and with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the concentrations 33, 100, 333, 1000, 2500 and 5000 µg/plate.
The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2021
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Arocalor
- Test concentrations with justification for top dose:
- Based on these observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 µg/mL in the presence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.
- Vehicle / solvent:
- Sterile water
- Untreated negative controls:
- yes
- Positive controls:
- yes
- Details on test system and experimental conditions:
- Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 63830396) with a doubling time approximately 12 -14 hours and modal chromosome number of 20 was used as the test system.
The cell line was tested for mycoplasma in the testing facility. The karyotype analysis of this cell line was periodically performed and documented.
Cells were grown in T- 25 cm2 and T-75 cm2 flasks at 37 ± 1 °C in a carbon dioxide incubator (5 ± 0.2 % CO2 in air) - Evaluation criteria:
- Metaphases of all concentrations of the test item, the positive and vehicle control cultures were scored.
- Statistics:
- Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The test item, C.I. Pigment Yellow 175 was not clastogenic in CHO-K1 cells at the tested concentration and under the conditions of testing employed.
- Executive summary:
The clastogenic potential of the test item, C.I. Pigment Yellow 175 to induce chromosomal aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO-K1) cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).
The study consisted of a preliminary cytotoxicity test and a chromosome aberration assay. Chromosome aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.
The test item formed a free flowing suspension in sterile water at 200 mg/mL and a pasty suspension in DMSO at 200 mg/mL. Hence, sterile water was selected as the vehicle of choice for the chromosomal aberration test.
In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, the test item did not exhibit the required level of cytotoxicity (reduction in the cell growth by 55± 5 % of the concurrent vehicle control)up to the highest tested concentration of 250µg/mL,both, in the presence and absence of metabolic activation (short term treatment) and in the absence of metabolic activation (long term treatment).
Based on these observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 µg/mL in thepresence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.
In the chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate at the concentrations of 15.63, 62.5 and 250mg/mL in Experiments 1, 2 and 3 of the chromosomal aberration assay. Concurrent vehicle (SW) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation at 21 hour exposure) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at approximately 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.
At the highest concentrations tested, the reduction in cell growth as RICC was 47, 43 and 53 % in experiments 1, 2 and 3, respectively, compared to the vehicle control.
A total of 300 metaphases each from the SW control, each treatment level and the positive controls were evaluated for chromosomal aberrations.
There was no evidence of statistically significant induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.
The study indicated that the test item, C.I. Pigment Yellow 175 was not clastogenic at the concentrations tested and under the conditions of testing
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 2 NOV 2004 to 27 DEC 2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD TG 476)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
RPMI 1640 (Hepes buffered medium) supplemented with 15% horse serum, penicillin/streptomycin (100 U/mL respectively 100 µl/mL), 220 µg/mL sodium pyruvate and 1.25 U/mL Amphotericin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 (induced with phenobarbital/beta-naphtoflavone)
- Test concentrations with justification for top dose:
- Experiment I:
without S9 mix: 11.7; 23.4; 46.9; 93.8; 187.5, and 375.0 microgram/mL
with S9 mix: 23.4; 46.9; 93.8; 187.5, and 375.0 microgram/mL
Experiment II: without S9 mix: 11.7; 23.4; 46.9; 93.8; and 187.5 microgram/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent and its relative non-toxicity to the cell cultures - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in suspension
DURATION
- Exposure duration: 4 h (Experiment I: with and w/o metabolic activation) and 24 h (Experiment II w/o metabolic activation)
- Expression time (cells in growth medium): 3 days total (i.e. experiment I: 4 hours treatment period plus 68 hours expression time; experiment II: 24 hours treatment period plus 48 hours expression time)
- Selection time (if incubation with a selection agent): 10 to 15 days
SELECTION AGENT (mutation assays): trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: two independent experiments, using two parallel cultures each
NUMBER OF CELLS USED: Per culture 1 x 10EXP7 cells
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other: relative suspension growth - Evaluation criteria:
- A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.
A test item producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
The test item is classified as mutagenic if it reproducibly induces a mutation frequency that is at least two times higher than the spontaneous mutation frequency (negative or solvent control) in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.
Results of test groups are rejected if the relative total growth, the relative suspension growth and/or cloning efficiency 1 (survival of the cells after treatment) is less than 10 % of the solvent control or the cloning efficiency 2 (viability) after the expression period is less than 20 %.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. - Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- indicated by reduced relative cloning efficiency 1 in experiment I at 375 µg/mL in both cultures
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- experiment I: 1st culture at 375 µg/mL, 2nd culture at 187.5 µg/mL and above; experiment II: both cultures at 187.5 µg/mL and above
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Precipitation of the test item was observed by the unaided eye at 187.5 and 375.0 microgram/mL in all experimental parts.
COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequency did not exceed the historical range of negative and solvent controls at any concentration with and without metabolic activation. The threshold of two times the number of mutant colonies/10EXP6 cells was reached at 46.9 microgram/mL in the second culture of experiment I (with S9 mix). Since this effect was not observed at higher concentrations or in the parallel culture, this isolated effect was judged as biologically irrelevant.
The mutation frequency of the positive control in the first culture of experiment I fell just short of the historical range of positive controls with 233 versus 292 colonies per 10EXP6 cells. Since the threshold of twice the mutation frequency of the solvent control was clearly exceeded and the mean value of both positive controls is well within the historical range, this effect was considered as irrelevant fluctuation.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I without metabolic activation relevant toxicity (relative cloning efficiency 1 and / or relative total growth of less than 50 % survival) was detected at 187.5 µg/mL and above in the second culture. In culture I a clear toxic effect occurred at the highest concentration of 375 µg/mL.
In experiment I with metabolic activation a toxic effect indicated by a reduced relative cloning efficiency 1 was observed at 375 µg/mL in both cultures.
In the second experiment (24 h treatment without metabolic activation) relevant toxic effects occurred at 187.5 µg/mL and above in both cultures. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results (migrated information):
negative
The test substance did not increase the mutant frequency at the TK+/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test. - Executive summary:
Mouse Lymphoma cells (L5178Y TK+/-) were treated with the test substance at five or six concentrations in the range of 11.7 to 375 microgram/mL. Precipitation of the test substance was detectable at dose level 187.5 and 375 microgram/mL. The solvent control gave mutant frequencies within the normal range. The positive controls gave marked increases in the mutant frequency, both in the presence or absence of metabolic activation, indicating the satisfactory performance of the test system. The test substance demonstrated no statistically significant or dose related increases in mutant frequency at any dose level, either with or without metabolic activation, thus indicating that the test item is not mutagenic under the conditions tested.
- 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:
- From 2 NOV 2004 to 27 DEC 2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD TG 476)
- Justification for type of information:
- See read across justfication document in chapter 13
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
RPMI 1640 (Hepes buffered medium) supplemented with 15% horse serum, penicillin/streptomycin (100 U/mL respectively 100 µl/mL), 220 µg/mL sodium pyruvate and 1.25 U/mL Amphotericin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 (induced with phenobarbital/beta-naphtoflavone)
- Test concentrations with justification for top dose:
- Experiment I:
without S9 mix: 11.7; 23.4; 46.9; 93.8; 187.5, and 375.0 microgram/mL
with S9 mix: 23.4; 46.9; 93.8; 187.5, and 375.0 microgram/mL
Experiment II: without S9 mix: 11.7; 23.4; 46.9; 93.8; and 187.5 microgram/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent and its relative non-toxicity to the cell cultures - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in suspension
DURATION
- Exposure duration: 4 h (Experiment I: with and w/o metabolic activation) and 24 h (Experiment II w/o metabolic activation)
- Expression time (cells in growth medium): 3 days total (i.e. experiment I: 4 hours treatment period plus 68 hours expression time; experiment II: 24 hours treatment period plus 48 hours expression time)
- Selection time (if incubation with a selection agent): 10 to 15 days
SELECTION AGENT (mutation assays): trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: two independent experiments, using two parallel cultures each
NUMBER OF CELLS USED: Per culture 1 x 10EXP7 cells
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other: relative suspension growth - Evaluation criteria:
- A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.
A test item producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
The test item is classified as mutagenic if it reproducibly induces a mutation frequency that is at least two times higher than the spontaneous mutation frequency (negative or solvent control) in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.
Results of test groups are rejected if the relative total growth, the relative suspension growth and/or cloning efficiency 1 (survival of the cells after treatment) is less than 10 % of the solvent control or the cloning efficiency 2 (viability) after the expression period is less than 20 %.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. - Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- indicated by reduced relative cloning efficiency 1 in experiment I at 375 µg/mL in both cultures
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- experiment I: 1st culture at 375 µg/mL, 2nd culture at 187.5 µg/mL and above; experiment II: both cultures at 187.5 µg/mL and above
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Precipitation of the test item was observed by the unaided eye at 187.5 and 375.0 microgram/mL in all experimental parts.
COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequency did not exceed the historical range of negative and solvent controls at any concentration with and without metabolic activation. The threshold of two times the number of mutant colonies/10EXP6 cells was reached at 46.9 microgram/mL in the second culture of experiment I (with S9 mix). Since this effect was not observed at higher concentrations or in the parallel culture, this isolated effect was judged as biologically irrelevant.
The mutation frequency of the positive control in the first culture of experiment I fell just short of the historical range of positive controls with 233 versus 292 colonies per 10EXP6 cells. Since the threshold of twice the mutation frequency of the solvent control was clearly exceeded and the mean value of both positive controls is well within the historical range, this effect was considered as irrelevant fluctuation.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I without metabolic activation relevant toxicity (relative cloning efficiency 1 and / or relative total growth of less than 50 % survival) was detected at 187.5 µg/mL and above in the second culture. In culture I a clear toxic effect occurred at the highest concentration of 375 µg/mL.
In experiment I with metabolic activation a toxic effect indicated by a reduced relative cloning efficiency 1 was observed at 375 µg/mL in both cultures.
In the second experiment (24 h treatment without metabolic activation) relevant toxic effects occurred at 187.5 µg/mL and above in both cultures. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results (migrated information):
negative
The test substance did not increase the mutant frequency at the TK+/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test. - Executive summary:
Mouse Lymphoma cells (L5178Y TK+/-) were treated with the test substance at five or six concentrations in the range of 11.7 to 375 microgram/mL. Precipitation of the test substance was detectable at dose level 187.5 and 375 microgram/mL. The solvent control gave mutant frequencies within the normal range. The positive controls gave marked increases in the mutant frequency, both in the presence or absence of metabolic activation, indicating the satisfactory performance of the test system. The test substance demonstrated no statistically significant or dose related increases in mutant frequency at any dose level, either with or without metabolic activation, thus indicating that the test item is not mutagenic under the conditions tested.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2021
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- See read across justification document in chapter 13
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Arocalor
- Test concentrations with justification for top dose:
- Based on these observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 µg/mL in the presence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.
- Vehicle / solvent:
- Sterile water
- Untreated negative controls:
- yes
- Positive controls:
- yes
- Details on test system and experimental conditions:
- Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 63830396) with a doubling time approximately 12 -14 hours and modal chromosome number of 20 was used as the test system.
The cell line was tested for mycoplasma in the testing facility. The karyotype analysis of this cell line was periodically performed and documented.
Cells were grown in T- 25 cm2 and T-75 cm2 flasks at 37 ± 1 °C in a carbon dioxide incubator (5 ± 0.2 % CO2 in air) - Evaluation criteria:
- Metaphases of all concentrations of the test item, the positive and vehicle control cultures were scored.
- Statistics:
- Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The test item, C.I. Pigment Yellow 175 was not clastogenic in CHO-K1 cells at the tested concentration and under the conditions of testing employed.
- Executive summary:
The clastogenic potential of the test item, C.I. Pigment Yellow 175 to induce chromosomal aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO-K1) cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).
The study consisted of a preliminary cytotoxicity test and a chromosome aberration assay. Chromosome aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.
The test item formed a free flowing suspension in sterile water at 200 mg/mL and a pasty suspension in DMSO at 200 mg/mL. Hence, sterile water was selected as the vehicle of choice for the chromosomal aberration test.
In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, the test item did not exhibit the required level of cytotoxicity (reduction in the cell growth by 55± 5 % of the concurrent vehicle control)up to the highest tested concentration of 250µg/mL,both, in the presence and absence of metabolic activation (short term treatment) and in the absence of metabolic activation (long term treatment).
Based on these observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 µg/mL in thepresence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.
In the chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate at the concentrations of 15.63, 62.5 and 250mg/mL in Experiments 1, 2 and 3 of the chromosomal aberration assay. Concurrent vehicle (SW) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation at 21 hour exposure) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at approximately 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.
At the highest concentrations tested, the reduction in cell growth as RICC was 47, 43 and 53 % in experiments 1, 2 and 3, respectively, compared to the vehicle control.
A total of 300 metaphases each from the SW control, each treatment level and the positive controls were evaluated for chromosomal aberrations.
There was no evidence of statistically significant induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.
The study indicated that the test item, C.I. Pigment Yellow 175 was not clastogenic at the concentrations tested and under the conditions of testing
- 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:
- From JUN 16 2011 to 26 JUL 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: minimal essential medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbital / beta-naphtoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- With metabolic activation:
Experiment I: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0
Experiment II: 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0
Without metabolic activation:
Experiment I: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0
Experiment II: 0.2, 0.7, 2.1, 6.2, 18.5, 55.6, 166.7, 500.0 - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of test item - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation
- 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 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium (minimal essential medium)
DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 18 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: about 1.5
NUMBER OF CELLS EVALUATED: at least 100 per culture, except for the positive control in Experiment I without metabolic activation, where only 50 metaphases were evaluated.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell numbers
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes - Evaluation criteria:
- Evaluation of the cultures was performed according to the OECD Guideline using NIKON microscopes with 100x objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) and relative cell numbers were determined.
In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases) - Statistics:
- Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
- Key result
- 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
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The highest treatment concentration in this study, 500.0 µg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment I, visible precipitation of the test item in the culture medium was observed at 15.6 µg/mL and above in the absence and presence of S9 mix at the end of treatment. In Experiment II precipitation was observed at 6.2 µg/mL and above in the absence of S9 mix and at 12.5 µg/mL and above in the presence of S9 mix at the end of treatment.
No relevant influence on osmolarity or pH value was observed. The osmolarity and pH-value were determined in the solvent control and the maximum concentration of Experiment I and II without metabolic activation:
Exp. Solvent control Test item 500.0 µg/mL
I Osmolarity (mOsm) 380 390
pH-value 7.3 7.3
II Osmolarity (mOsm) 395 392
pH-value 7.5 7.4
No cytotoxicity indicated by reduced cell numbers or mitotic indices were observed after treatment with the test item .
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 3.3 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.0 - 2.5 % aberrant cells, excluding gaps) and within the range of the laboratory’s historical control data: 0.0 - 4.0 % aberrant cells, excluding gaps.
In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.3 - 3.9 %) as compared to the rates of the solvent controls (1.3 - 3.6 %).
In both experiments, either EMS (600 or 1000 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line), when tested up to precipitating concentrations. - Remarks on result:
- other: strain/cell type: V79 cells
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations. - Executive summary:
The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:
Without S9 mix
With S9 mix
Exp. I
Exp. II
Exp. I
Exp. II
Exposure period
4 hrs
18 hrs
4 hrs
4 hrs
Recovery
14 hrs
-
14 hrs
14 hrs
Preparation interval
18 hrs
18 hrs
18 hrs
18 hrs
In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment I without metabolic activation, where only 50 metaphases were evaluated.
The highest applied concentration (500.0 µg/mL, approx. 1.4 mM) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.
Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. The evaluated experimental points and the results are summarised in Table1.
In the absence and presence of S9 mix no cytotoxicity was observed up to the highest evaluated concentration, where test item precipitation occurred.
No clastogenicity was observed at the concentrations evaluated, either with or without metabolic activation.
No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.
No relevant increase in endomitotic cells was found after treatment with the test item as compared to the frequencies of the control cultures.
Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2020
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: In vitro mammalian cell gene mutation test using the Hprt gene
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1,
(ATCC CCL-61, Lot 4765275) - Metabolic activation:
- with and without
- Untreated negative controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- Details on test system and experimental conditions:
- Cells were grown in tissue culture flasks at 37 ± 1 °C in a humidified carbon dioxide incubator (5 ± 0.2 % CO2 in air)
- Rationale for test conditions:
- Test approaches currently accepted under the OECD for the assessment of mammalian cell gene mutation involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.
Established CHO cell line is useful in in vitro gene mutation testing because it is easily cultured in standard medium, has a small number of large chromosomes each with a more or less distinctive morphology and a relatively short cycle time - Evaluation criteria:
- CRITERIA FOR ACCEPTABILITY OF THE TEST
The assay will be considered valid if the following criteria are met:
a) The concurrent vehicle control data is within the range of the laboratory historical control data.
b) The concurrent positive control substances should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent vehicle control.
c) Two experimental conditions are tested unless one results in positive response.
d) Adequate number of cells and analyzable concentrations are tested under each of the experimental conditions.
e) The criteria for the selection of top concentration are consistent with those described in the guideline.
EVALUATION AND INTERPRETATION OF RESULTS
When all the validity criteria are fulfilled:
1. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• The increase is concentration-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
2 A test chemical is considered to be clearly negative if, in all experimental conditions examined:
• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- It is concluded that the test item, C.I.Pigment Yellow 194 does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.
- Executive summary:
The genotoxic potential of the test item C.I.Pigment Yellow 194 induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.
The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).
C.I.Pigment Yellow 194 was insoluble in sterile water and formed a free flowing suspension in Dimethyl sulfoxide (DMSO) at
200 mg/mL.In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 27 and 32 % at the
1000 µg/mL, in the presence and absence of metabolic activation, respectively. There was precipitation of the test item in the test medium at and above
1000 µg/mL, both in the presence and absence of metabolic activation. There was no appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 1500 µg/mL was tested in the gene mutation assay.In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 23.44, 93.75, 375 and 1500 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.
There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.
The results of the forward gene mutation test at thehprtlocus with C.I.Pigment Yellow 194 indicated that the test item was non-mutagenic under the conditions of this study
- 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:
- From JUN 16 2011 to 26 JUL 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- see read across justification in chapter 13
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: minimal essential medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbital / beta-naphtoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- With metabolic activation:
Experiment I: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0
Experiment II: 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0
Without metabolic activation:
Experiment I: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0
Experiment II: 0.2, 0.7, 2.1, 6.2, 18.5, 55.6, 166.7, 500.0 - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of test item - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation
- 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 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium (minimal essential medium)
DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 18 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: about 1.5
NUMBER OF CELLS EVALUATED: at least 100 per culture, except for the positive control in Experiment I without metabolic activation, where only 50 metaphases were evaluated.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell numbers
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes - Evaluation criteria:
- Evaluation of the cultures was performed according to the OECD Guideline using NIKON microscopes with 100x objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) and relative cell numbers were determined.
In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases) - Statistics:
- Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
- Key result
- 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
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The highest treatment concentration in this study, 500.0 µg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment I, visible precipitation of the test item in the culture medium was observed at 15.6 µg/mL and above in the absence and presence of S9 mix at the end of treatment. In Experiment II precipitation was observed at 6.2 µg/mL and above in the absence of S9 mix and at 12.5 µg/mL and above in the presence of S9 mix at the end of treatment.
No relevant influence on osmolarity or pH value was observed. The osmolarity and pH-value were determined in the solvent control and the maximum concentration of Experiment I and II without metabolic activation:
Exp. Solvent control Test item 500.0 µg/mL
I Osmolarity (mOsm) 380 390
pH-value 7.3 7.3
II Osmolarity (mOsm) 395 392
pH-value 7.5 7.4
No cytotoxicity indicated by reduced cell numbers or mitotic indices were observed after treatment with the test item .
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 3.3 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.0 - 2.5 % aberrant cells, excluding gaps) and within the range of the laboratory’s historical control data: 0.0 - 4.0 % aberrant cells, excluding gaps.
In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.3 - 3.9 %) as compared to the rates of the solvent controls (1.3 - 3.6 %).
In both experiments, either EMS (600 or 1000 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line), when tested up to precipitating concentrations. - Remarks on result:
- other: strain/cell type: V79 cells
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations. - Executive summary:
The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:
Without S9 mix
With S9 mix
Exp. I
Exp. II
Exp. I
Exp. II
Exposure period
4 hrs
18 hrs
4 hrs
4 hrs
Recovery
14 hrs
-
14 hrs
14 hrs
Preparation interval
18 hrs
18 hrs
18 hrs
18 hrs
In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment I without metabolic activation, where only 50 metaphases were evaluated.
The highest applied concentration (500.0 µg/mL, approx. 1.4 mM) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.
Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. The evaluated experimental points and the results are summarised in Table1.
In the absence and presence of S9 mix no cytotoxicity was observed up to the highest evaluated concentration, where test item precipitation occurred.
No clastogenicity was observed at the concentrations evaluated, either with or without metabolic activation.
No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.
No relevant increase in endomitotic cells was found after treatment with the test item as compared to the frequencies of the control cultures.
Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2020
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- See read across justification document in chapter 13
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: In vitro mammalian cell gene mutation test using the Hprt gene
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1,
(ATCC CCL-61, Lot 4765275) - Metabolic activation:
- with and without
- Untreated negative controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- Details on test system and experimental conditions:
- Cells were grown in tissue culture flasks at 37 ± 1 °C in a humidified carbon dioxide incubator (5 ± 0.2 % CO2 in air)
- Rationale for test conditions:
- Test approaches currently accepted under the OECD for the assessment of mammalian cell gene mutation involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.
Established CHO cell line is useful in in vitro gene mutation testing because it is easily cultured in standard medium, has a small number of large chromosomes each with a more or less distinctive morphology and a relatively short cycle time - Evaluation criteria:
- CRITERIA FOR ACCEPTABILITY OF THE TEST
The assay will be considered valid if the following criteria are met:
a) The concurrent vehicle control data is within the range of the laboratory historical control data.
b) The concurrent positive control substances should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent vehicle control.
c) Two experimental conditions are tested unless one results in positive response.
d) Adequate number of cells and analyzable concentrations are tested under each of the experimental conditions.
e) The criteria for the selection of top concentration are consistent with those described in the guideline.
EVALUATION AND INTERPRETATION OF RESULTS
When all the validity criteria are fulfilled:
1. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• The increase is concentration-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
2 A test chemical is considered to be clearly negative if, in all experimental conditions examined:
• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- It is concluded that the test item, C.I.Pigment Yellow 194 does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.
- Executive summary:
The genotoxic potential of the test item C.I.Pigment Yellow 194 induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.
The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).
C.I.Pigment Yellow 194 was insoluble in sterile water and formed a free flowing suspension in Dimethyl sulfoxide (DMSO) at
200 mg/mL.In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 27 and 32 % at the
1000 µg/mL, in the presence and absence of metabolic activation, respectively. There was precipitation of the test item in the test medium at and above
1000 µg/mL, both in the presence and absence of metabolic activation. There was no appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 1500 µg/mL was tested in the gene mutation assay.In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 23.44, 93.75, 375 and 1500 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.
There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.
The results of the forward gene mutation test at thehprtlocus with C.I.Pigment Yellow 194 indicated that the test item was non-mutagenic under the conditions of this study
Referenceopen allclose all
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
The test item precipitated in the overlay agar at the following concentrations (µg/plate):
Strain |
Experiment I |
Experiment II |
||
|
without S9 mix |
with S9 mix |
without S9 mix |
with S9 mix |
TA 1535 |
/ |
1000 - 5000 |
333 - 5000 |
333 - 5000 |
TA 1537 |
/ |
1000 - 5000 |
33 - 5000 |
333 - 5000 |
TA 98 |
/ |
1000 - 5000 |
333 - 5000 |
333 - 5000 |
TA 100 |
/ |
1000 |
333 - 5000 |
333 - 5000 |
WP2 uvrA |
/ |
1000 - 5000 |
333 - 5000 |
333 - 5000 |
/ no visible precipitation observed
The undissolved particles had no influence on the data recording.
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
The test item precipitated in the overlay agar at the following concentrations (µg/plate):
Strain |
Experiment I |
Experiment II |
||
|
without S9 mix |
with S9 mix |
without S9 mix |
with S9 mix |
TA 1535 |
/ |
1000 - 5000 |
333 - 5000 |
333 - 5000 |
TA 1537 |
/ |
1000 - 5000 |
33 - 5000 |
333 - 5000 |
TA 98 |
/ |
1000 - 5000 |
333 - 5000 |
333 - 5000 |
TA 100 |
/ |
1000 |
333 - 5000 |
333 - 5000 |
WP2 uvrA |
/ |
1000 - 5000 |
333 - 5000 |
333 - 5000 |
/ no visible precipitation observed
The undissolved particles had no influence on the data recording.
Table 1: Summary of results of the chromosome aberration study with the test substance
Exp. |
Preparation |
Test item |
Endomitotic |
Polyploid |
Cell numbers |
Mitotic indices |
Aberrant cells |
||
|
interval |
concentration |
cells |
cells |
in % |
in % |
in % |
||
|
|
in µg/mL |
in % |
in % |
of control |
of control |
incl. gaps* |
excl. gaps* |
with exchanges |
Exposure period 4 hrs without S9 mix |
|||||||||
I |
18 hrs |
Solvent control1 |
0.0 |
3.6 |
100.0 |
100.0 |
1.0 |
1.0 |
0.0 |
|
|
Positive control2# |
n.d. |
n.d. |
n.d. |
91.0 |
33.0 |
33.0S |
20.0 |
|
|
3.9 |
0.0 |
3.4 |
82.0 |
95.3 |
0.5 |
0.5 |
0.5 |
|
|
7.8 |
0.0 |
3.0 |
115.6 |
100.0 |
0.5 |
0.5 |
0.5 |
|
|
15.6P |
0.0 |
2.7 |
92.7 |
111.4 |
1.5 |
1.0 |
0.0 |
Exposure period 18 hrs without S9 mix |
|||||||||
II |
18 hrs |
Solvent control1 |
0.0 |
2.4 |
100.0 |
100.0 |
3.0 |
2.0 |
0.0 |
|
|
Positive control3 |
n.d. |
n.d. |
n.d. |
71.2 |
18.0 |
17.5S |
10.0 |
|
|
0.7 |
0.0 |
2.7 |
106.6 |
101.8 |
1.0 |
1.0 |
0.0 |
|
|
2.1 |
0.0 |
1.9 |
76.4 |
119.6 |
1.0 |
1.0 |
0.0 |
|
|
6.2P |
0.0 |
1.9 |
75.7 |
83.2 |
2.5 |
2.0 |
0.0 |
Exposure period 4 hrs with S9 mix |
|||||||||
I |
18 hrs |
Solvent control1 |
0.1 |
3.6 |
100.0 |
100.0 |
1.5 |
1.0 |
0.5 |
|
|
Positive control4 |
n.d. |
n.d. |
n.d. |
87.0 |
14.0 |
13.0S |
4.5 |
|
|
3.9 |
0.1 |
2.4 |
79.3 |
115.1 |
1.5 |
1.5 |
0.0 |
|
|
7.8 |
0.1 |
3.6 |
78.9 |
92.5 |
3.0 |
2.5 |
0.5 |
|
|
15.6P |
0.3 |
3.9 |
113.1 |
102.5 |
2.5 |
2.0 |
0.5 |
II |
18 hrs |
Solvent control1 |
0.0 |
2.3 |
100 |
100 |
2.5 |
2.5 |
0.0 |
|
|
Positive control4 |
n.d. |
n.d. |
n.d. |
71.4 |
16.0 |
14.5S |
7.0 |
|
|
3.1## |
0.0 |
1.9 |
111.9 |
78.3 |
4.0 |
3.3 |
1.3 |
|
|
6.3## |
0.0 |
2.0 |
90.5 |
99.2 |
3.0 |
3.0 |
0.8 |
|
|
12.5P |
0.0 |
1.3 |
76.8 |
112.0 |
2.5 |
2.5 |
2.5 |
* Inclusive cells carrying exchanges
# Evaluation of 50 metaphases per culture
## Evaluation of 200 metaphases per culture
n.d. Not determined
P Precipitation occurred at the end of treatment
S Aberration frequency statistically significant higher than corresponding control values
1 DMSO 0.5 % (v/v)
2 EMS 1000.0 µg/mL
3 EMS 600.0 µg/mL
4 CPA 1.4 µg/mL
Table 1: Summary of results of the chromosome aberration study with the test substance
Exp. |
Preparation |
Test item |
Endomitotic |
Polyploid |
Cell numbers |
Mitotic indices |
Aberrant cells |
||
|
interval |
concentration |
cells |
cells |
in % |
in % |
in % |
||
|
|
in µg/mL |
in % |
in % |
of control |
of control |
incl. gaps* |
excl. gaps* |
with exchanges |
Exposure period 4 hrs without S9 mix |
|||||||||
I |
18 hrs |
Solvent control1 |
0.0 |
3.6 |
100.0 |
100.0 |
1.0 |
1.0 |
0.0 |
|
|
Positive control2# |
n.d. |
n.d. |
n.d. |
91.0 |
33.0 |
33.0S |
20.0 |
|
|
3.9 |
0.0 |
3.4 |
82.0 |
95.3 |
0.5 |
0.5 |
0.5 |
|
|
7.8 |
0.0 |
3.0 |
115.6 |
100.0 |
0.5 |
0.5 |
0.5 |
|
|
15.6P |
0.0 |
2.7 |
92.7 |
111.4 |
1.5 |
1.0 |
0.0 |
Exposure period 18 hrs without S9 mix |
|||||||||
II |
18 hrs |
Solvent control1 |
0.0 |
2.4 |
100.0 |
100.0 |
3.0 |
2.0 |
0.0 |
|
|
Positive control3 |
n.d. |
n.d. |
n.d. |
71.2 |
18.0 |
17.5S |
10.0 |
|
|
0.7 |
0.0 |
2.7 |
106.6 |
101.8 |
1.0 |
1.0 |
0.0 |
|
|
2.1 |
0.0 |
1.9 |
76.4 |
119.6 |
1.0 |
1.0 |
0.0 |
|
|
6.2P |
0.0 |
1.9 |
75.7 |
83.2 |
2.5 |
2.0 |
0.0 |
Exposure period 4 hrs with S9 mix |
|||||||||
I |
18 hrs |
Solvent control1 |
0.1 |
3.6 |
100.0 |
100.0 |
1.5 |
1.0 |
0.5 |
|
|
Positive control4 |
n.d. |
n.d. |
n.d. |
87.0 |
14.0 |
13.0S |
4.5 |
|
|
3.9 |
0.1 |
2.4 |
79.3 |
115.1 |
1.5 |
1.5 |
0.0 |
|
|
7.8 |
0.1 |
3.6 |
78.9 |
92.5 |
3.0 |
2.5 |
0.5 |
|
|
15.6P |
0.3 |
3.9 |
113.1 |
102.5 |
2.5 |
2.0 |
0.5 |
II |
18 hrs |
Solvent control1 |
0.0 |
2.3 |
100 |
100 |
2.5 |
2.5 |
0.0 |
|
|
Positive control4 |
n.d. |
n.d. |
n.d. |
71.4 |
16.0 |
14.5S |
7.0 |
|
|
3.1## |
0.0 |
1.9 |
111.9 |
78.3 |
4.0 |
3.3 |
1.3 |
|
|
6.3## |
0.0 |
2.0 |
90.5 |
99.2 |
3.0 |
3.0 |
0.8 |
|
|
12.5P |
0.0 |
1.3 |
76.8 |
112.0 |
2.5 |
2.5 |
2.5 |
* Inclusive cells carrying exchanges
# Evaluation of 50 metaphases per culture
## Evaluation of 200 metaphases per culture
n.d. Not determined
P Precipitation occurred at the end of treatment
S Aberration frequency statistically significant higher than corresponding control values
1 DMSO 0.5 % (v/v)
2 EMS 1000.0 µg/mL
3 EMS 600.0 µg/mL
4 CPA 1.4 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
The test item did not cause mutagenic or clastogenic effects in vitro in bacteria as well as mammalian cells
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
