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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: - | CAS number: 1309389-73-8
- 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
In a key Bacterial Reverse Mutation Assay according to OECD guideline 471, the results indicate that the test item, under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E.coli strain WP2 uvr A in the presence and absence of a metabolic activation.
In a key in vitro Mammalian Cell Micronucleus Test according to OECD guideline 487, the test item did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9), when tested up to cytotoxic concentrations.
According to a valid QSAR prediction using DEREK Nexus v. 6.2, no mutagenic properties of the test item were estimated.
Overall conclusion: The test substance is neither mutagenic nor clastogenic in two in vitro genotoxicity tests and a QSAR prediction.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- (Q)SAR
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
- Justification for type of information:
- Please refer to the QMRF and QPRF files provided under the section attached justification.
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- Estimates the mutagenic properties of chemicals using structural alert relationships.
- Specific details on test material used for the study:
- SMILES: O=C(C=Cc2ccc1OCOc1c2)N(c3ccccc3)c4ccccc4
- Key result
- Species / strain:
- other: no alerts
- Remarks on result:
- no mutagenic potential (based on QSAR/QSPR prediction)
- Conclusions:
- Using Derek Nexus 6.2.0, no mutagenic properties of the test item were estimated.
- Executive summary:
Mutagenicity was estimated using Derek Nexus 6.2.0. No mutagenic properties were estimated based on the described QSAR method (Derek, 2017).
The adequacy of a prediction depends on the following conditions:
a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;
b) the (Q)SAR model is applicable to the query chemical
c) the (Q)SAR result is reliable
d) the (Q)SAR model is relevant for the regulatory purpose.
For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.
Description of the prediction Model
The prediction model was described using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file.
Assessment of estimation domain
The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2013-08-22 to 2014-02-14
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 2010
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes: human peripheral blood lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Human peripheral blood lymphocytes from two healthy, non-smoking female volunteers
- Suitability of cells: Yes, blood was received from donors who were not suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors were non smokers and not heavy drinkers of alcohol. Donors were not taking any form of medication (contraceptive pill excluded).
- Normal cell cycle time: The measured cell cycle time of the donors falls within the range of 13±2 hours.
For lymphocytes:
- Sex, age and number of blood donors: Blood from 2 female donors was used in the range-finding and main study. The age of the donors was 28 and 31 in the range-finding study and 30 and 31 in the main experiment.
- Whether whole blood or separated lymphocytes were used: Whole blood cultures were used
- Whether blood from different donors were pooled or not: Blood was pooled using equal volumes from each donor prior to use.
- Mitogen used for lymphocytes: Phytohaemagglutinin (PHA, reagent grade)
MEDIA USED
- Type and composition of media: HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin
- Temperature: 37±1°C - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- Source of S9: Obtained from Molecular Toxicology Incorporated, USA where it is prepared from male Sprague Dawley rats induced with Aroclor 1254.
- Method of preparation of S9 mix: Glucose-6-phosphate (180 mg/mL), β-Nicotinamide adenine dinucleotide phosphate (25 mg/mL), Potassium chloride (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2.
- Concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of the S9 mix in the test system was 5%.The final concentration of S9 in the test system was 2%.
- Quality controls of S9: Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities) as specified in the Certificate of Analysis. - Test concentrations with justification for top dose:
- Range-finding study: 1.814 to 500 µg/mL (3-hour treatment +/- S9 and 20-hour treatment -S9);
Main study: 5 to 100 µg/mL (3-hour treatment -S9), 10 to 100 µg/mL (3-hour treatment +S9) and 2 to 40 µg/mL (20-hour treatment -S9)
Concentrations that were evaluated for micronuclei were: 10, 30 and 60 µg/mL (3-hour treatment -S9), 10, 30, 55 and 60 µg/mL (3-hour treatment +S9) and 8, 14 and 20 µg/mL (20-hour treatment -S9) - Vehicle / solvent:
- - Vehicles used: DMSO (test substance, CPA), purified water (VIN, MMC)
- Justification for choice of solvent/vehicle: Solubility properties. DMSO is a commonly used solvent for this type of assay with a broad historical control database.
- Justification for percentage of solvent in the final culture medium: The maximum solvent concentration in the final culture medium was 1% which is known from historical control data to be not toxic and not mutagenic in this type of assay. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO (test substance, CPA), purified water (VIN, MMC)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- vinblastine
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: For the range-finding study: Single (test substance) and duplicate (solvent control); for the main experiment: Duplicate (test substance and positive control) and quadruplicate (solvent control)
- Number of independent experiments: 1 range-finding assay and 1 main assay (each consisting of 3 tested exposure conditions)
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in: Medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours (+/- S9) and 20 hours (- S9)
- Harvest time after the end of treatment (sampling/recovery times): 24 hours for all test conditions. For the 3-hour treatments, cytochalasin B was added after the 3-hour treatment period and incubated for further 21 hours. For the 20-hour treatment, cytochalasin B was added at the beginning od the treatment period and incubated, together with the test substance for 24 hours.
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Identity of cytokinesis blocking substance, concentration, and duration and period of cell exposure: Cytochalasin B was added (0.1 mL per culture) to the 24-hour treatment culture at the time of treatment. Cytochalasin B was added (0.1 mL per culture) to the 3-hour treatment cultures directly after the treatment period. The final concentration was 6 μg/mL per culture.
- Methods of slide preparation and staining technique used including the stain used: Fixed lymphocytes were centrifuged and resuspended in a minimal amount of fresh fixative. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried then stored protected from light at room temperature prior to staining. Slides were stained by immersion in 125 μg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 seconds, washed with PBS (with agitation) for a few seconds before transfer and immersion in a second container of PBS for approx. 10 minutes. Slides were air-dried and stored protected from light at room temperature prior to analysis.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Slides from the cytotoxicity range-finder experiment were examined for
proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. In the main experiment, slides were examined for RI to a minimum of 500 cells per culture.1000 binucleate cells from each culture (2000 per concentration) were analysed for micronuclei.
- Criteria for scoring micronucleated cells: See "Any other information on materials and methods incl. tables"
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Replication index
METHODS FOR MEASUREMENTS OF GENOTOXICIY
The proportion of binucleate cells with micronuclei (MNBN) as compared to the proportion in vehicle controls - Rationale for test conditions:
- Test concentrations were chosen based on a dose-range finding test.
3-hour treatment (-S9): Precipitation occured at concentration of 38.88 µg/mL and above. At this concentration, 51% cytotoxicity was reached.
3-hour treatment (+S9): Precipitation occured at concentration of 38.88 µg/mL and above. At this concentration, 41% cytotoxicity was reached. At 64.80 µg/mL, 57% cytotoxicity was reached.
24-hour treatment (-S9): Marked cytotoxicity occured at concentrations of 8.398 µg/mL (36%), 14 µg/mL (79%) and 23.33 µg/mL (100%). Precipitation occured at concentrations of 38.88 µg/mL and above.
On this basis, test concentrations for the main assay were selected. The highest concentration selected for micronucleus analysis following all treatment conditions induced 55±5% cytotoxicity. Slides from the highest selected concentration and two or three lower concentrations were taken for microscopic analysis, such that a range of cytotoxicity from maximum to little was covered. - Evaluation criteria:
- For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed. The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result - Statistics:
- The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportion of MNBN cells for each treatment condition was compared with the proportion in vehicle controls by using Fisher's exact test.
Probability values of p≤0.05 were accepted as significant. Additionally, the number of micronuclei per binucleate cell were obtained and recorded. - Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- The limit cytotoxicity of 55±5% was reached at 60 µg/mL (3-hour treatment with and without S9) and at 20 µg/mL (24-hour treatment without S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No marked changes in pH were observed at the highest concentration tested (500.0 μg/mL) in the Range Finder Experiment as compared to the concurrent vehicle controls.
- Data on osmolality: No marked changes in osmolality were observed at the highest concentration tested (500.0 μg/mL) in the Range Finder Experiment as compared to the concurrent vehicle controls (individual data not reported).
- Water solubility: The test item was soluble in DMSO up to a concentration of 175.0 mg/mL.
- Precipitation and time of the determination: Precipitation occured at the beginning of treatment from 38.88 µg/L onwards under all test conditions. Precipitation occured at the end of tretment from 108 and 180 µg/mL onwards for the 3-hour treatment times (-/+S9). Precipitation occured at harvest from 64.80 µg/mL (3-hours, -S9), from 108 µg/mL (3-hours, +S9) and from 38.88 µg/mL (24-hours, -S9) onwards under all test conditions.
- Definition of acceptable cells for analysis: See "Any other information on materials and methods incl. tables"
RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity was tested in a range-finding study at concentrations between 1.814 to 500 µg/mL in duplicate (vehicle control) or single cultures (test item) under all three incubation conditions (3-hour treatment with and without S9 and 24-hour treatment without S9). Dose-dependent cytotoxic effects were observed in all cultures (see "Rationale for test conditions"). On this basis, test concentrations for the main experiment were selected.
STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: No concentration-response relationship was observed.
- Statistical analysis: No statistical significance was observed for any of the the test substance concentrations and negative controls. Positive control yielded the expected statistically significant responses.
Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: See "Attached background material"
o In the case of the cytokinesis-block method: CBPI or RI; distribution of mono-, bi- and multi-nucleated cells: See "Attached background material"
- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate: See "Attached background material"
HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material" - Conclusions:
- In an in vitro Mammalian Cell Micronucleus Test according to OECD guideline 487, the test item did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9), when tested up to cytotoxic concentrations.
- Executive summary:
The test item was tested in an in vitro micronucleus assay according to OECD guideline 487 and GLP using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest concentrations used in the Micronucleus Experiment, were determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of the test item on the replication index (RI). Micronuclei were analysed at three or four concentrations and a summary of the data is presented in the following table:
Treatment
Concentration
(µg/mL)
Cytotoxicity
(%)
Mean
MNBN Cell
Frequency
(%)
Historical
Control
Range (%)
Statistical
Significance
3+21 hour -S9
Vehicle
-
0.80
0.10 – 1.10
-
10.00
11
1.05
NS
30.00
27
0.80
NS
60.00
55
0.65
NS
MMC, 0.80
ND
7.35
p ≤0.001
3+21 hour +S9
Vehicle
-
0.80
0.20 – 1.20
-
10.00
7
0.90
NS
30.00
27
0.85
NS
55.00
57
0.40
NS
60.00
50
0.45
NS
CPA, 6.25
ND
2.90
p ≤0.001
24+0 hour -S9
Vehicle
-
1.10
0.10 – 1.50
-
8.00
10
1.25
NS
14.00
24
1.00
NS
20.00
55
1.05
NS
VIN, 0.02
ND
16.92
p ≤0.001
Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in the vehicle cultures fell within, or very close to, the 95th percentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals, respectively, in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9.
Cells receiving these were sampled in the Micronucleus Experiment at 24 hours after the start of treatment; all compounds induced statistically significant increases in the proportion of cells with micronuclei. All acceptance criteria were considered met and the study was therefore accepted as valid. Treatment of cells with the test item in the absence and presence of S-9 resulted in frequencies of MNBN cells that were generally similar to and not significantly higher than those observed in concurrent vehicle controls for all concentrations analysed under all three treatment conditions. The MNBN cell frequency of all treated cultures fell within normal ranges with the exception of a single culture at 10 μg/mL, the lowest concentration analysed following 3+21 hours treatment in the presence of S-9. This isolated marginal increase of 1.3% compared to the normal range of 0.2-1.2% was not reproduced in the replicate culture at 10 g/mL or at any other concentration tested. In addition, as the mean MNBN cell frequency fell within the normal range and was not statistically significantly higher than the concurrent vehicle controls, it was considered of no biological relevance.
It is concluded that the test item did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9), when tested up to cytotoxic concentrations.
- 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:
- 2009-08-18 to 2009-09-25
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP guideline study - expiration date missing
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 1997-07-21
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- signed 2005-10-21
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
E. coli WP2 uvrA: trp- - 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:
- S9 fraction prepared from livers of male Wistar rats orally receiving phenobarbitone/β-naphthoflavone on three consecutive days
- Test concentrations with justification for top dose:
- Standard plate test (experiment 1): 0, 22, 110, 550, 2750 and 5500 μg/plate (with and without metabolic activation)
Preincubation test (experiment 2): 0, 22, 110, 550, 2750, and 5500 μg/plate (with and without metabolic activation) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to the limited solubility of the test item in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- Positive control with metabolic activation: concentrations: 2.5 μg/plate (strains TA 1535, TA 100, TA 1537, & TA 98) & 60 μg/plate (strain E. coli WP2 uvrA)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: N-methyl-N'-nitro-N-nitrosoguanidine
- Remarks:
- Positive control without metabolic activation: concentration: 5 μg/plate (strains TA 1535 & TA 100)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylenediamine
- Remarks:
- Positive control without metabolic activation: concentration: 10 μg/plate (strain TA 98)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Positive control without metabolic activation: concentration: 100 μg/plate (strain TA 1537)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- Positive control without metabolic activation: concentration: 5 μg/plate (strain E. coli WP2 uvrA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Experiment 1: in agar (plate incorporation); Experiment 2: preincubation
DURATION
- Preincubation period (experiment 2): about 20 minutes
- Exposure duration (experiments 1 & 2): 48 -72 hours at 37 °C
NUMBER OF REPLICATIONS (experiments 1 & 2): 3
DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer
is recorded for all test groups both with and without S9 mix in all experiments.
Precipitation of the test material is recorded. - Evaluation criteria:
- The test chemical is considered positive in this assay if the following criteria are met:
- a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test item is generally considered non-mutagenic in this test if:
- the number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other. - Statistics:
- no data
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- please refer to the field "Additional information on results" below
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- please refer to the field "Additional information on results" below
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- please refer to the field "Additional information on results" below
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- please refer to the field "Additional information on results" below
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True 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:
- cytotoxicity
- Remarks:
- please refer to the field "Additional information on results" below
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- STANDARD PLATE TEST (experiment 1):
No increase in the number of his+ or trp+ revertants was observed with and without metabolic activation.
PREINCUBATION TEST (experiment 2):
No increase in the number of his+ or trp+ revertants was observed with and without metabolic activation.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test (experiment 1) depending on the strain and test conditions at the highest applied concentration (5500 μg/plate).
In the preincubation assay (experiment 2) bacteriotoxicity (slight decrease in the number of his+ revertants, slight reduction in the titer) was occasionally observed depending on the strain and test conditions from about 2750 μg/plate onward.
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: test item precipitation was found from 110 μg/plate onward with and without S9 mix.
Please also refer to the field "Attached background material" below. - Conclusions:
- In a Bacterial Reverse Mutation Assay according to OECD guideline 471, the results indicate that the test item, under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E.coli strain WP2 uvr A in the presence and absence of a metabolic activation.
- Executive summary:
A bacterial reverse mutation assay was performed with the substance according to the OECD guideline 471 (1997) using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 as well as E. coli WP2 uvr A. The study was carried out using the standard plate test (experiment 1) and the preincubation test (experiment 2). Both experiments were conducted with and without metabolic activation and each concentration was plated in triplicate. The substance was tested at the following concentrations in the both experiments: 22, 110, 550, 2750, and 5500 µg/plate. Additionally, a vehicle control and several positive controls were run concurrently.
In both experiments no increase in the number of his+ or trp+ revertants was observed with and without metabolic activation.
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions at the highest applied concentration (5500 μg/plate). In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants, slight reduction in the titer) was occasionally observed depending on the strain and test conditions from about 2750 μg/plate onward. Test item precipitation was found from 110 µg/plate onward with and without metabolic activation.
The results of the negative as well as the positive controls performed corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
- the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;
- the (Q)SAR model is applicable to the query chemical
- the (Q)SAR result is reliable
- the (Q)SAR model is relevant for the regulatory purpose.
Key, Bacterial Reverse Mutation Assay, RL1
A bacterial reverse mutation assay was performed with the substance according to the OECD guideline 471 (1997) and GLP using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 as well as E. coli WP2 uvr A. The study was carried out using the standard plate test (experiment 1) and the preincubation test (experiment 2). Both experiments were conducted with and without metabolic activation and each concentration was plated in triplicate. The substance was tested at the following concentrations in the both experiments: 22, 110, 550, 2750, and 5500 µg/plate. Additionally, a vehicle control and several positive controls were run concurrently. In both experiments no increase in the number of his+ or trp+ revertants was observed with and without metabolic activation. A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions at the highest applied concentration (5500 μg/plate). In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants, slight reduction in the titer) was occasionally observed depending on the strain and test conditions from about 2750 μg/plate onward. Test item precipitation was found from 110 µg/plate onward with and without metabolic activation. The results of the negative as well as the positive controls performed corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.
Key, In Vitro Mammalian Cell Micronucleus Test, RL1
The test item was tested in an in vitro micronucleus assay according to OECD guideline 487 and GLP using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest concentrations used in the Micronucleus Experiment, were determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of the test item on the replication index (RI). Micronuclei were analysed at three or four concentrations and a summary of the data is presented in the following table:
Treatment | Concentration (µg/mL) | Cytotoxicity (%) | Mean MNBN Cell Frequency (%) | Historical Control Range (%) | Statistical Significance |
3+21 hour -S9 | Vehicle | - | 0.80 | 0.10 – 1.10 | - |
10.00 | 11 | 1.05 |
| NS | |
30.00 | 27 | 0.80 |
| NS | |
60.00 | 55 | 0.65 |
| NS | |
MMC, 0.80 | ND | 7.35 |
| p ≤0.001 | |
3+21 hour +S9 | Vehicle | - | 0.80 | 0.20 – 1.20 | - |
10.00 | 7 | 0.90 |
| NS | |
30.00 | 27 | 0.85 |
| NS | |
55.00 | 57 | 0.40 |
| NS | |
60.00 | 50 | 0.45 |
| NS | |
CPA, 6.25 | ND | 2.90 |
| p ≤0.001 | |
24+0 hour -S9 | Vehicle | - | 1.10 | 0.10 – 1.50 | - |
8.00 | 10 | 1.25 |
| NS | |
14.00 | 24 | 1.00 |
| NS | |
20.00 | 55 | 1.05 |
| NS | |
VIN, 0.02 | ND | 16.92 |
| p ≤0.001 |
Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in the vehicle cultures fell within, or very close to, the 95th percentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals, respectively, in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9.
Cells receiving these were sampled in the Micronucleus Experiment at 24 hours after the start of treatment; all compounds induced statistically significant increases in the proportion of cells with micronuclei. All acceptance criteria were considered met and the study was therefore accepted as valid. Treatment of cells with the test item in the absence and presence of S-9 resulted in frequencies of MNBN cells that were generally similar to and not significantly higher than those observed in concurrent vehicle controls for all concentrations analysed under all three treatment conditions. The MNBN cell frequency of all treated cultures fell within normal ranges with the exception of a single culture at 10 μg/mL, the lowest concentration analysed following 3+21 hours treatment in the presence of S-9. This isolated marginal increase of 1.3% compared to the normal range of 0.2-1.2% was not reproduced in the replicate culture at 10 g/mL or at any other concentration tested. In addition, as the mean MNBN cell frequency fell within the normal range and was not statistically significantly higher than the concurrent vehicle controls, it was considered of no biological relevance.
It is concluded that the test item did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9), when tested up to cytotoxic concentrations.
QSAR, In Vitro Gene Mutation Study in Mammalian Cells, RL2
Mutagenicity was estimated using Derek Nexus 6.2.0. No mutagenic properties were estimated based on the described QSAR method (Derek, 2017).
The adequacy of a prediction depends on the following conditions:
For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to the endpoint study record.
Description of the prediction Model
The prediction model was described using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file.
Assessment of estimation domain
The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.
Overall conclusion: The test substance is neither mutagenic nor clastogenic in two in vitro genotoxicity tests and a QSAR prediction.
Justification for classification or non-classification
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified and labelled according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.
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.