Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 230-258-1 | CAS number: 6992-11-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
PB25
The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.
The test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.
RA from PR176
The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.
The test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.
The test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.
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:
- DEC 2004 to JAN 2005
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD TG 471), with Prival modification for azo-dyes perfrormed under GLP
- 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
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not applicable
- 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: plate incorporation 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 up to 72 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 as an increase is not considered biologically relevant. - Statistics:
- Arithmetic means and standard deviation of the counted colonies were calculated.
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- COMPARISON WITH HISTORICAL CONTROL DATA:
The number of colonies was slightly above our historical control range in strains TA 1537 (negative and solvent control, exp. II) and TA 98 (solvent control, exp. II) with S9 mix. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both experiments.
Minor toxic effects, evident as a reduction in the number of revertants, occurred at 5000 µg/plate in experiment II in strains TA 1537 (without S9 mix) and TA 98 (with and without S9 mix).
Precipitation occured in experiment II starting from 1000 µg/plate. - 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 plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both experiments.
Minor toxic effects, evident as a reduction in the number of revertants, occurred at 5000 µg/plate in experiment II in strains TA 1537 (without S9 mix) and TA 98 (with and without S9 mix).
Precipitation occured in experiment II starting from 1000 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
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 01 SEP 2011 to 25 OCT 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD 473) and GLP
- 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:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbital/beta-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- With metabolic activation:
Experiment I: 1.0, 1.7, 3.0, 5.2, 9.1, 16.0, 28.0, 49.0, 85.7, 150.0 µg/mL
Experiment II: 1.0, 1.7, 3.0, 5.2, 9.1, 16.0, 28.0 µg/mL
Without metabolic activation:
Experiment I: 1.0, 1.7, 3.0, 5.2, 9.1, 16.0, 28.0, 49.0, 85.7, 150.0 µg/mL
Experiment II: 1.0, 1.7, 3.0, 5.2, 9.1, 16.0, 28.0, 49.0, 85.7, 150.0 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473 - 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 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium
DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: about 1.5
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - Evaluation criteria:
- Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion 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. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. - Statistics:
- Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The test item, suspended in acetone, was assessed for its potential to induce chromosomal aberrations in human lymphocytes 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 S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after the start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 150.0 µg/mL (approx. 0.3 mM) 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 9.1 µg/mL and above in the absence and presence of S9 mix. In addition, precipitation occurred in Experiment II, at 9.1 µg/mL and above in the absence of S9 mix and at 3.0 µg/mL and above in the presence of S9 mix. No relevant influence on osmolarity or pH value was observed.
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. At higher concentrations severe test item precipitation on the slides was observed.
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 (0.0 - 3.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 1.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (550 or 770 µg/mL) and CPA (7.5 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. - Conclusions:
- Interpretation of results: negative
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes 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 acetone, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitroin two independent experiments. The following study design was performed:
Without S9 mix
With S9 mix
Exp. I
Exp. II
Exp. I & II
Exposure period
4 hrs
22 hrs
4 hrs
Recovery
18 hrs
-
18 hrs
Preparation interval
22 hrs
22 hrs
22 hrs
In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations.
The highest applied concentration in this study (150.0 µg/mL of the test item, approx. 0.3 mM) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.
Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473.
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. At higher concentrations severe test item precipitation on the slides was observed.
In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to 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
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD 476) and GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
- Test concentrations with justification for top dose:
- Experiment I:
without metabolic activation (4 hours treatment): 6.3; 12.5; 25.0; 50.0; 100.0; and 200.0 µg/mL
with metabolic activation (4 hours treatment): : 6.3; 12.5; 25.0; 50.0; 100.0; and 200.0 µg/mL
Experiment II:
without metabolic activation (24 hours treatment): 6.3; 12.5; 25.0; 50.0; 100.0; and 200.0 µg/mL
with metabolic activation (4 hours treatment): : 6.3; 12.5; 25.0; 50.0; 100.0; and 200.0 µg/mL
In experiment I and II the concentration of 200 µg/mL with and without metabolic activation was not continued to avoid analysis of too many precipitating concentrations. - Vehicle / solvent:
- deionised water
- 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:
- 7,12-dimethylbenzanthracene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days
SELECTION AGENT (mutation assays): 6-Thioguanine
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1,5x10exp. 6
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
- 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
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected
- Effects of osmolality: Not increased
- Evaporation from medium: Not examined
- Precipitation: In the pre-experiment increasing precipitation occurred from the lowest to the highest test item concentration. In experiment I precipitation was observed at 12.5 µg/mL and above without metabolic activation, and at 25.0 µg/mL and above with metabolic activation. In experiment II precipitation was observed at 25.0 µg/mL and above with and without metabolic activation.
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 3200 µg/mL limited by the solubility of the test item in DMSO and aqueous medium. Test item concentrations between 25 µg/mL and 3200 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant cytotoxic effects indicated by a relative suspension growth below 50 % were observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 25 µg/mL and above in the presence and absence of metabolic activation (4 hours treat-ment). Following continuous treatment (24 hours) without metabolic activation, precipita-tion was observed at 50.0 µg/mL and above.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
COMPARISON WITH HISTORICAL CONTROL DATA: Complies
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effects, indicated by a relative cloning efficiency I (survival) of less than 50% compared to the corresponding solvent control occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. - Conclusions:
- Interpretation of results: negative
In conclusion it can be stated that under the experimental conditions reported, the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay. - Executive summary:
The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.
The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. Due to bacterial contamination, the first experiment with metabolic activation was terminated prematurely and repeated. The data of the repeat experiment are reported as experiment I with metabolic activation.
The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The maximum concentration of the main experiments was limited by the solubility of the test item. Precipitation at the end of treatment, visible to the unaided eye occurred in the first experiment at 12.5 µg/mL and above without metabolic activation and at 25.0 µg/mL and above in the presence of metabolic activation. In the second experiment precipitation occurred at 25.0 µg/mL and above with and without metabolic activation.
No relevant cytotoxic effects, indicated by a relative cloning efficiency I (survival) of less than 50% compared to the corresponding solvent control occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The induction factor did not reach or exceed the threshold of three times the corresponding solvent control at any concentration.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.2 up to 21.7 mutants per 106cells; the range of the groups treated with the test item was from 4.1 up to 36.3 mutants per 106cells.
(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- see Section 13 for the attached Justification for Read-across report
- Reason / purpose for cross-reference:
- read-across source
- 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
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results: negative
In conclusion it can be stated that under the experimental conditions reported, the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay. - Executive summary:
The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.
The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. Due to bacterial contamination, the first experiment with metabolic activation was terminated prematurely and repeated. The data of the repeat experiment are reported as experiment I with metabolic activation.
The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The maximum concentration of the main experiments was limited by the solubility of the test item. Precipitation at the end of treatment, visible to the unaided eye occurred in the first experiment at 12.5 µg/mL and above without metabolic activation and at 25.0 µg/mL and above in the presence of metabolic activation. In the second experiment precipitation occurred at 25.0 µg/mL and above with and without metabolic activation.
No relevant cytotoxic effects, indicated by a relative cloning efficiency I (survival) of less than 50% compared to the corresponding solvent control occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The induction factor did not reach or exceed the threshold of three times the corresponding solvent control at any concentration.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.2 up to 21.7 mutants per 106cells; the range of the groups treated with the test item was from 4.1 up to 36.3 mutants per 106cells.
(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
Referenceopen allclose all
Summary of results of the chromosomal aberration study
Exp. |
Preparation |
Test item |
Mitotic indices |
Aberrant cells |
|
|||||
|
interval |
concentration |
in % |
in % |
|
|||||
|
|
in µg/mL |
of control |
incl. gaps* |
excl. gaps* |
carrying exchanges |
|
|||
|
Exposure period 4 hrs without S9 mix |
|
||||||||
I |
22 hrs |
Solvent control1 |
100.0 |
1.0 |
1.0 |
0.0 |
|
|||
|
|
Positive control2 |
99.5 |
12.0 |
11.5S |
0.5 |
|
|||
|
|
3.0 |
90.3 |
3.5 |
3.0 |
0.0 |
|
|||
|
|
5.2 |
88.7 |
1.0 |
1.0 |
0.0 |
|
|||
|
|
9.1P |
104.1 |
0.5 |
0.5 |
0.0 |
|
|||
|
Exposure period 22 hrs without S9 mix |
|||||||||
II |
22 hrs |
Solvent control1 |
100.0 |
2.0 |
0.5 |
0.0 |
|
|||
|
|
Positive control3 |
50.9 |
13.0 |
12.0S |
1.5 |
|
|||
|
|
3.0 |
100.4 |
0.5 |
0.5 |
0.0 |
|
|||
|
|
5.2 |
87.8 |
2.5 |
2.0 |
0.0 |
|
|||
|
|
9.1P |
98.2 |
2.5 |
2.5 |
0.0 |
|
|||
|
Exposure period 4 hrs with S9 mix |
|||||||||
I |
22 hrs |
Solvent control1 |
100.0 |
0.5 |
0.5 |
0.0 |
|
|||
|
|
Positive control4 |
47.7 |
13.0 |
11.5S |
1.0 |
|
|||
|
|
3.0 |
96.2 |
1.5 |
1.0 |
0.0 |
|
|||
|
|
5.2 |
95.8 |
1.0 |
1.0 |
0.0 |
|
|||
|
|
9.1P |
77.6 |
1.5 |
1.5 |
0.0 |
|
|||
II |
22 hrs |
Solvent control1 |
100.0 |
1.5 |
1.5 |
0.0 |
|
|||
|
|
Positive control5 |
41.7 |
17.0 |
16.5S |
2.0 |
|
|||
|
|
1.0 |
98.0 |
2.5 |
2.5 |
0.0 |
|
|||
|
|
1.7 |
99.5 |
1.0 |
1.0 |
0.0 |
|
|||
|
|
3.0P |
70.4 |
0.0 |
0.0 |
0.0 |
|
|||
* Including cells carrying exchanges
P Precipitation occurred at the end of treatment
S Aberration frequency statistically significant higher than corresponding control values
1 Acetone 0.5 % (v/v)
2 EMS 770.0 µg/mL
3 EMS 550.0 µg/mL
4 CPA 7.5 µg/mL
5 CPA 15.0 µg/mL
Summary Table
| relative | relative | relative | mutant | relative | relative | relative | mutant | ||||||
| conc. | P | S9 | cloning | cell | cloning | colonies/ | induction | cloning | cell | cloning | colonies/ | induction | |
| µg/mL | mix | efficiency I | density | efficiency II | 106cells | factor | efficiency I | density | efficiency II | 106cells | factor | ||
| % | % | % | % | % | % | ||||||||
| Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| Experiment I / 4 h treatment | culture I | culture II | |||||||||||
| Solvent control with water | - | 100.0 | 100.0 | 100.0 | 12.7 | 1.0 | 100.0 | 100.0 | 100.0 | 12.5 | 1.0 | ||
| Positive control (EMS) | 150.0 | - | 69.9 | 99.0 | 112.3 | 89.7 | 7.1 | 61.4 | 109.7 | 104.3 | 87.8 | 7.0 | |
| Test item | 6.3 | - | 103.2 | 142.9 | 104.0 | 6.7 | 0.5 | 98.5 | 88.2 | 113.1 | 4.8 | 0.4 | |
| Test item | 12.5 | P | - | 97.7 | 119.7 | 103.6 | 14.5 | 1.1 | 97.3 | 96.2 | 103.0 | 10.0 | 0.8 |
| Test item | 25.0 | P | - | 98.2 | 107.5 | 111.1 | 6.6 | 0.5 | 93.9 | 101.3 | 105.5 | 11.8 | 0.9 |
| Test item | 50.0 | P | - | 89.6 | 121.1 | 111.2 | 12.9 | 1.0 | 94.5 | 89.8 | 104.0 | 8.0 | 0.6 |
| Test item | 100.0 | P | - | 91.8 | 116.2 | 109.0 | 4.3 | 0.3 | 87.6 | 93.6 | 101.6 | 14.7 | 1.2 |
| Test item | 200.0 | P | - | 88.0 | culture was not continued# | 82.0 | culture was not continued# | ||||||
| Solvent control with water | + | 100.0 | 100.0 | 100.0 | 8.4 | 1.0 | 100.0 | 100.0 | 100.0 | 8.2 | 1.0 | ||
| Positive control (DMBA) | 1.1 | + | 61.2 | 69.7 | 78.6 | 296.8 | 35.5 | 66.7 | 61.0 | 93.9 | 273.5 | 33.2 | |
| Test item | 6.3 | + | 87.3 | 110.7 | 78.7 | 10.7 | 1.3 | 93.3 | 85.4 | 92.4 | 10.8 | 1.3 | |
| Test item | 12.5 | + | 87.7 | 101.4 | 99.7 | 5.2 | 0.6 | 91.3 | 104.8 | 75.0 | 13.3 | 1.6 | |
| Test item | 25.0 | P | + | 86.6 | 87.4 | 91.0 | 7.5 | 0.9 | 93.8 | 101.4 | 94.6 | 4.1 | 0.5 |
| Test item | 50.0 | P | + | 97.0 | 102.8 | 75.9 | 5.5 | 0.7 | 86.1 | 105.9 | 86.4 | 8.5 | 1.0 |
| Test item | 100.0 | P | + | 88.9 | 96.8 | 97.4 | 11.0 | 1.3 | 88.4 | 115.1 | 98.0 | 14.3 | 1.7 |
| Test item | 200.0 | P | + | 87.2 | culture was not continued# | 91.5 | culture was not continued# | ||||||
| Experiment II / 24 h treatment | culture I | culture II | |||||||||||
| Solvent control with water | - | 100.0 | 100.0 | 100.0 | 21.7 | 1.0 | 100.0 | 100.0 | 100.0 | 19.1 | 1.0 | ||
| Positive control (EMS) | 150.0 | - | 83.2 | 115.8 | 81.9 | 422.0 | 19.4 | 73.8 | 105.8 | 66.8 | 436.7 | 22.9 | |
| Test item | 6.3 | - | 103.7 | 115.1 | 89.9 | 22.9 | 1.1 | 91.1 | 110.9 | 100.6 | 15.3 | 0.8 | |
| Test item | 12.5 | - | 100.0 | 76.4 | 87.9 | 13.0 | 0.6 | 96.1 | 111.6 | 91.4 | 6.6 | 0.3 | |
| Test item | 25.0 | P | - | 92.1 | 104.7 | 86.0 | 22.1 | 1.0 | 89.7 | 78.8 | 92.4 | 19.6 | 1.0 |
| Test item | 50.0 | P | - | 98.2 | 128.2 | 88.8 | 13.4 | 0.6 | 93.7 | 89.4 | 95.9 | 12.0 | 0.6 |
| Test item | 100.0 | P | - | 98.3 | 148.7 | 102.0 | 15.5 | 0.7 | 87.2 | 106.6 | 89.8 | 13.4 | 0.7 |
| Test item | 200.0 | P | - | 101.0 | culture was not continued# | 97.2 | culture was not continued# | ||||||
| Experiment II / 4 h treatment | |||||||||||||
| Solvent control with water | + | 100.0 | 100.0 | 100.0 | 15.6 | 1.0 | 100.0 | 100.0 | 100.0 | 12.9 | 1.0 | ||
| Positive control (DMBA) | 1.1 | + | 51.7 | 141.4 | 72.2 | 927.5 | 59.5 | 61.6 | 86.4 | 75.9 | 629.2 | 48.8 | |
| Test item | 6.3 | + | 104.1 | 103.2 | 91.3 | 17.5 | 1.1 | 106.9 | 95.2 | 91.5 | 12.9 | 1.0 | |
| Test item | 12.5 | + | 99.9 | 103.3 | 99.1 | 36.3 | 2.3 | 97.3 | 88.9 | 100.6 | 9.2 | 0.7 | |
| Test item | 25.0 | P | + | 101.2 | 102.2 | 102.1 | 14.9 | 1.0 | 98.7 | 87.7 | 103.2 | 5.6 | 0.4 |
| Test item | 50.0 | P | + | 97.7 | 111.8 | 98.8 | 20.1 | 1.3 | 105.2 | 95.9 | 99.2 | 15.2 | 1.2 |
| Test item | 100.0 | P | + | 87.8 | 108.0 | 84.6 | 12.8 | 0.8 | 87.8 | 94.2 | 99.2 | 7.6 | 0.6 |
| Test item | 200.0 | P | + | 92.6 | culture was not continued# | 93.7 | culture was not continued# | ||||||
# culture not continued to avoid evaluation of too many precipitating concentrations
P precipitation
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 were observed
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.
