Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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.
Diss Factsheets
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: 252-346-9 | CAS number: 35074-77-2
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Link to relevant study records
- 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:
- 23 Jan 2015 - 23 Apr 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP-compliant guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS) and 1% (v/v) penicillin/streptomycin and 1% (v/v) amphotericine B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbital and β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- 1st Experiment
without S9 mix: 0; 6.3; 12.5; 25.0; 50.0; 100.0 μg/mL
with S9 mix: 0; 6.3; 12.5; 25.0; 50.0; 100.0 μg/mL
2nd Experiment
without S9 mix: 0; 5.0; 10.0; 20.0; 40.0; (80.0) μg/mL
with S9 mix: 0; 5.0; 10.0; 20.0; 40.0; (80.0) μg/mL
Numbers in parantheses were discontinued - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, acetone was selected as vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 2 weeks
SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: duplicate cultures were used for each of the two experiments
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10exp6 clonable cells) or isolated statistically significant increases without a doseresponse relationship may indicate a biological effect but are not regarded as sufficien evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantl increased above the concurrent negative control and is within our historical negative control data range. - Statistics:
- An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced by test substance treatment
- Effects of osmolality: not influenced by test substance treatment
- Precipitation: In the 1st Experiment in the absence and presence of S9 mix, test substance precipitation was observed in culture medium at the end of treatment at 50 μg/mL and above and in the 2nd Experiment at 40 μg/mL and above.
RANGE-FINDING/SCREENING STUDIES:
In the pretest the parameters pH value and osmolarity were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, suspension of the test substance in the vehicle acetone was observed at 2600 μg/mL and above. In culture medium, test substance precipitation occurred by the end of treatment at concentrations of 40.6 μg/mL and above in the absence and presence of S9 mix. After 4 hours treatment in the presence and absence of S9 mix, cytotoxicity was not observed as indicated by a reduced relative cloning efficiency of about or below 20% relative survival.
COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments after 4 hours treatment with the test substance the values for the corrected mutation frequencies were clearly within the range of our historical negative control data. The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies were clearly within our historical positive control data
range.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective vehicle control values were observed in both experiments in the absence and presence of S9 mix. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation. - Executive summary:
The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested.
Test groups printed in bold type were evaluated in this study:
1st Experiment
without S9 mix: 0; 6.3; 12.5; 25.0; 50.0; 100.0 μg/mL
with S9 mix: 0; 6.3; 12.5; 25.0; 50.0; 100.0 μg/mL
2nd Experiment
without S9 mix: 0; 5.0; 10.0; 20.0; 40.0; 80.0 μg/mL
with S9 mix: 0; 5.0; 10.0; 20.0; 40.0; 80.0 μg/mL
Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6-thioguaninecontaining medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations. In this study in the absence and the presence of metabolic activation, no cytotoxicity was observed up to the highest tested concentration evaluated for gene mutations. Based on the results of the present study, the test substance either did not cause any relevant increase in the mutant frequencies without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Reference
Summary of Results
Exp. | Exposure period [h] |
Test groups [µg/mL] | S9 mix |
Prec.* | Genotoxicity** MFcorr.[per 106 cells] |
Cytotoxicity*** | |
CE1 [%] |
CE2 [%] |
||||||
1 | 4 | Vehicle control1 | - | n.d. | 3.57 | 100.0 | 100.0 |
6.3 | - | - | 4.48 | 94.8 | 98.2 | ||
12.5 | - | - | 0.95 | 99.3 | 95.7 | ||
25 | - | - | 0 | 100 | 91.2 | ||
50 | - | + | 0.88 | 89.3 | 96.7 | ||
100 | - | + | 1.19 | 94.4 | 97.6 | ||
Positive control2 | - | n.d. | 89.32 | 101.6 | 79.8 | ||
2 | 4 | Vehicle control1 | - | n.d. | 4.28 | 100.0 | 100.0 |
5 | - | - | 2.35 | 107.5 | 104.6 | ||
10 | - | - | 0 | 96.1 | 104.8 | ||
20 | - | - | 3.02 | 106.6 | 102.1 | ||
40 | - | + | 0.71 | 102.9 | 94.3 | ||
80 | - | + | n.c.1 | 98.8 | n.c.1 | ||
Positive control2 | - | n.d. | 148.58 | 96.1 | 89.5 | ||
1 | 4 | Vehicle control1 | + | n.d. | 1.02 | 100.0 | 100.0 |
6.3 | + | - | 1.67 | 92.8 | 103.9 | ||
12.5 | + | - | 1.91 | 99.5 | 108.2 | ||
25 | + | - | 0 | 108.2 | 103.9 | ||
50 | + | + | 1.67 | 103.4 | 101.7 | ||
100 | + | + | 3.13 | 101.5 | 108.3 | ||
Positive control3 | + | n.d. | 104.61 | 106 | 88.9 | ||
2 | 4 | Vehicle control1 | + | n.d. | 5.86 | 100.0 | 100.0 |
5 | + | - | 0 | 95.8 | 110.3 | ||
10 | + | - | 4.05 | 95.8 | 103.2 | ||
20 | + | - | 2.37 | 103.1 | 112.6 | ||
40 | + | + | 0 | 97.6 | 107.6 | ||
80 | + | + | n.c.1 | 99.8 | n.c.1 | ||
Positive control3 | + | n.d. | 233.23 | 89.5 | 99.1 |
* Precipitation in culture medium at the end of exposure period
** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value
*** Cloning efficiency related to the respective vehicle control
n.c.1 Culture was not continued since a minimum of only four analysable concentrations are required
n.d. Not determined
1 Acetone 1% (v/v)
2 EMS 400 μg/mL
3 DMBA 1.25 μg/mL
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Ames Test
In a reverse gene mutation assay in bacteria, four tester strains (S. typhimurium TA1535, TA1537, TA100, TA98) were exposed to the test substance at concentrations of 0.2, 2, 20, 200, 2000 µg/plate in the presence and absence of mammalian metabolic activation. Each concentration was tested in triplicate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background. The test substance was not mutagenic in the S. typhimurium strains TA1535, TA1537, TA100 and TA98 under the experimental conditions with or without metabolic activation (Batelle, 1978).
HPRT-Assay
In a GLP-compliant mammalian mutagenicity assay following OECD guideline 476, the test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses the dose rage used was 0 -100 µg/ml without, and 0 -40 µg/ml with S9 mix.
The test substance did not increase the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were close to the range of the concurrent vehicle control values and within the range of the historical negative control data. The mutation frequencies of the vehicle control groups were within the historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. In this study in the absence and the presence of metabolic activation, no cytotoxicity was observed up to the highest tested concentration evaluated for gene mutations. To summarize, in the absence and the presence of metabolic activation, the test article is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.
Dominant Lethal study
In an in vivo dominant lethal test, 20 male mice (Tif: MAGf) were orally treated with the test substance at doses of 0, 1000 and 3000 mg/kg bw. They were then mated to untreated females from the same strain over a period of six weeks. At the end of each week the females were replaced by new ones (2 females/male/week). The experiment was done to evaluate any cytotoxic or mutagenic effects on the male germinal cells as expressed by the loss of pre-implementation zygotes as well as by the rate deaths of post-implementation stages of embryonic development. The females mated to males which had been treated with the test substance did not differ significantly from the females mated to control, neither in mating ratio nor in the number of implantations and embryonic deaths (resorptions). No evidence of a dominant lethal effect was observed in the progeny of male mice treated with the test substance.
Assessment
The test article was not mutagenic in vitro and in vivo. In addition, when tested in life time feeding studies in rats, no evidence of carcinogenic activity was reported. Therefore, the test article is considered to be not genotoxic. No further testing is required.
Justification for selection of genetic toxicity endpoint
Evaluation of genetic toxicity was done based on the complete data set available.
Justification for classification or non-classification
Dangerous Substance Directive (67/548/EEC)
The available in vitro and in vivo studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance does not need to be classified and labelled for mutagenic toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EC.
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental in vitro and in vivo test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance does not need to be classified and labelled for mutagenic toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation EC No 605/2014.
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.