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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 203-686-1 | CAS number: 109-60-4
- 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
Micronucleus assay: OECD 487, GLP, TK6 cells, 34.38 – 1100.00 µg/ml (with and without S9), three experiments. No evidence of genotoxicity.
HPRT: OECD 476, GLP, CHO, 68.8 – 1100.00 µg/ml (with and without S9), four experiments. No evidence of genotoxicity.
Ames: Equivalent to 471, GLP, S. typhimurium TA100, TA 98, TA1535, TA1537, and TA1538, 100-10,000 µg/plate. No evidence of genotoxicity.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Jul - Dec 2017
- 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:
- Jul 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Commission Regulation (EC) No 640/2012; B.49
- Version / remarks:
- JUl 2012
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source: BASF SE Ludwigshafen, Germany
- Batch No.of test material: 83294456P0
- Date of production: 10 Dec 2016
- Purity: 99.9%
- Physical state, appearance: liquid, colorless, clear
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability was guaranteed until 08 Nov 2018
- Homogeneity: Homogeneity was guaranteed on account of the high purity and was ensured by mixing before preparation
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with the vehicle to achieve the required stock solution. The test substance preparation was shaken thoroughly. - Species / strain / cell type:
- human lymphoblastoid cells (TK6)
- Cytokinesis block (if used):
- CytB
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction obtained from phenobarbital and beta-naphthoflavone induced rat liver
- Test concentrations with justification for top dose:
- 1st experiment: 34.38, 68.75, 137.50, 275.00, 550.00, and 1100.00 µg/ml (with and without S9)
2nd experiment: 137.50, 275.00, 550.00, and 1100.00 µg/ml (with S9)
3rd experiment: 200.00, 400.00, 550.00, 800.00, and 1100.00 µg/ml (with and without S9)
A pretest was performed using 1100 µg/ml as top concentration. No cytotoxicity about or below 55% +/- 5% of control was observed. - Vehicle / solvent:
- - Vehicle used: RPMI 1640 (culture medium)
- Justification for choice of solvent: The test substance has a good solubility in water - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Vehicle control is negative control
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment I: 4 h (with and without S9), Experiment II: 4 h (with S9), Experiment III: 4 h (with S9) and 24 h (without S9)
- Recovery time: Experiment I and II: 20 h (with and without S9), Experiment III: 40 h (with S9)
- Harvest time: Experiment I and II: 24 h (with and without S9), Experiment III: 24 h (without S9) and 44 h (with S9)
SPINDLE INHIBITOR (cytogenetic assays): CytB (final concentration: 3 µg/ml; stock: 0.6 mg/ml in DMSO)
STAIN (for cytogenetic assays): 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/ml)
NUMBER OF REPLICATIONS: Two
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Slide preparation was carried out based on the method described by Fenech (1993). Single cell suspensions were prepared from each test group by resuspending. Then, the cell number per flask of each cell suspension was determined using a cell counter. Subsequently, 5x10^4 cells per slide were centrifuged at 600 rpm for 7 minutes onto labeled slides using a Cytospin centrifuge. At least two slides per flask were prepared. In the case of strongly reduced cell numbers no slides were prepared. After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes.
Before scoring, the slides were stained with a mixture of DAPI and propidium iodide in Fluoroshield™ at a concentration of 0.25 μg/mL each. By the use of the combination of both fluorescence dyes it can be differentiated between DNA (DAPI; excitation: 350 nm, emission: 460 nm) and cytoplasm (PI; excitation: 488 nm, emission: 590 nm).
NUMBER OF CELLS EVALUATED: As a rule, at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not specified
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The analysis of micronuclei was carried out following the criteria of Countryman and Heddle (1976):
− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only binucleated cells were scored.
DETERMINATION OF CYTOTOXICITY
- Method: reduced cell number, Cytokinesis-block proliferation index (CBPI)
OTHER EXAMINATIONS:
- pH value, osmolality, solubility - Evaluation criteria:
- Assessment criteria
A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data
A test substance is considered to be clearly negative if the following criterion is met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data - Statistics:
- The statistical evaluation of the data was carried out using an appropriate statistical analysis.
The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE).
If the results of this test were statistically significant compared with the respective vehicle control (p ≤ 0.05), labels (S) have been printed in the tables.
Furthermore, a statistical trend test (SAS; Proc Reg) was performed to assess a possible dose-related increase of micronucleated cells. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the micronuclei rate of each test group and the independent variable was the concentration. The trend was performed one-sided (increased dose-related trend) with a significande level of 0.05. - Species / strain:
- human lymphoblastoid cells (TK6)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced by test substance
- Effects of osmolality: not influenced by test substance
- Water solubility: yes
- Precipitation: no
- Definition of acceptable cells for analysis: Before use each batch was checked for mycoplasma contamination, karyotype stability, and growth characteris
HISTORICAL CONTROL DATA (with ranges)
- Positive historical control data: 2.0 - 6.5% micronucleated cells
- Negative (solvent/vehicle) historical control data: 0.1 - 1.3% micronucleated cells - Conclusions:
- Thus, under the experimental conditions chosen here, the conclusion is drawn that the test substance has not the potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in human TK6 cells in the absence and the presence of metabolic activation.
- Executive summary:
The test substance was assessed for its potential to induce micronuclei in TK6 cells in vitro (clastogenic or aneugenic activity). Three independent experiments were carried out 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. The test groups printed in bold type were evaluated.
1st Experiment
4 hours exposure, 24 hours harvest time, without S9 mix
0; 34.38; 68.75; 137.50; 275.00; 550.00 and 1100.00 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix (not valid)
0; 34.38; 68.75; 137.50; 275.00; 550.00 and 1100.00 μg/mL
2nd Experiment
4 hours exposure, 24 hours harvest time, with S9 mix
0; 137.50; 275.00; 550.00 and 1100.00 μg/mL
3rd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix
0; 200.00; 400.00; 550.00; 800.00 and 1100.00 μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix
0; 200.00; 400.00; 550.00; 800.00 and 1100.00 μg/mL
In the 1st Experiment cultures treated with the positive control in the presence of S9 mix did not show an increase in the micronucleus frequency. Thus, this experimental part is considered as invalid and was repeated in the 2nd Experiment. The results of the invalid part of the 1st Experiment will not be shown in this report. The raw data of this experimental part will be archived with the raw data of this study.
A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.
The negative controls gave frequencies of micronucleated cells within our historical negative control data range for TK6 cells. Both positive control substances, mitomycin C (MMC) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei.
Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in human TK6 cells in the absence and the presence of metabolic activation.
On the basis of the results of the present study, the test substance did not cause any
biologically relevant increase in the number of cells containing micronuclei either without S9
mix or after adding a metabolizing system.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Jan - Jun 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- Jul 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Commission Regulation (EC) No 440/2008; B.17
- Version / remarks:
- May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- Aug 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht
- Type of assay:
- other: HPRT
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source: BASF SE Ludwigshafen, Germany
- Batch No.of test material: 83294456P0
- Date of production: 10 Dec 2016
- Purity: 99.9%
- Physical state, appearance: liquid, colorless, clear
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability was guaranteed until 08 Nov 2018
- Homogeneity: Homogeneity was guaranteed on account of the high purity and was ensured by mixing before preparation
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with the vehicle to achieve the required stock solution. The test substance preparation was treated with ultrasonic waves thoroughly. - Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction obtained from phenobarbital and beta-naphthoflavone induced rats
- Test concentrations with justification for top dose:
- Experiment I: 68.8, 137.5, 275.0, 550.0, and 1100.0 µg/ml (with and without S9)
Experiment II: 87.5, 175.0, 350.0, 700.0, and 1100.0 µg/ml (with and without S9)
Experiment III: 87.5, 175.0, 350.0, 700.0, and 1100.0 µg/ml (with and without S9)
Experiment IV: 87.5, 175.0, 350.0, 700.0, and 1100.0 µg/ml (with S9)
Top concentration was established in a pretest. No cytotoxicity was observed as indicated by a reduced relative servival of about or below 20% of control. - Vehicle / solvent:
- - Vehicle used: Ham's F12 (culute medium)
- Justification for choice of vehicle: The test substance has a good solubility in water - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Solvent control is negative control
- 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
- Cell density at seeding: 20*10^6
DURATION
- Preincubation period: 20-24 h
- Exposure duration: 4 h
- 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 of cells): 5-7 days
SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):
NUMBER OF REPLICATIONS:
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
NUMBER OF CELLS EVALUATED:
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
OTHER EXAMINATIONS:
- pH, osmolality, solubility, cell morphology - Evaluation criteria:
- Assessment criteria
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative control value and the range of our laboratory’s historical negative control data (95% control limit). Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data - Statistics:
- An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report.
The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0.
In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction (1979). The calculation was performed using R.
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables.
However, both, biological and statistical significance are 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:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Without S9, pH was not influenced. With S9, there was a slight shift in the pH values towards an acid pH value in the highest applied concentrations. However, the increased pH values did not influence the results on cytotoxicity and genotoxicity of this study.
- Effects of osmolality: not influenced by test substance
- Water solubility: yes
- Precipitation: no
- Definition of acceptable cells for analysis:
- Cell morphology: After 4 h treatment, neither with nor without S9, cell morphology and attachment of cells was not adversely influenced in any test group.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: 42.47-419.90 (without S9), 21.52-270.48 (with S9)
- Negative (solvent/vehicle) historical control data: 0.00-6.49 (without S9), 0.00-7.43 (with S9) - Conclusions:
- 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.
- 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. Four independent experiments were carried out, with and/or 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 top concentration was determined to be the limit concentration of approx. 10 mM (1100 μg/mL). In the main experiments the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:
1st Experiment
without S9 mix
0; 68.8; 137.5; 275.0; 550.0; 1100.0 μg/mL
with S9 mix
0; 68.8; 137.5; 275.0; 550.0; 1100.0 μg/mL
2nd Experiment (not valid; data not shown)
without S9 mix
0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL
with S9 mix
0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL
3rd Experiment
without S9 mix
0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL
with S9 mix (not valid; data not shown)
0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL
4th Experiment
with S9 mix
0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL
Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week.
Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The negative controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.
In this study, in all experiments, in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.
Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in all experiments performed independently of each other.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline with acceptable restrictions (Purity unknown, missing E.coli/TA 102 strain to detect crosslinking and oxidising agents; therefore, reliability score "1" was not adopted from OECD SIDS)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- E. coli/ TA102 strain is missing (former OECD guideline 472)
- Principles of method if other than guideline:
- according to Ames B.N. et al., Mutat. Res., 31. 347-364, (1975)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his-
- Species / strain / cell type:
- other: S. typhimurium TA100, TA 98, TA1535, TA1537, and TA1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- Arochlor 1254-induced rat liver S-9 mix
- Test concentrations with justification for top dose:
- 0, 100, 333, 1000, 3333, 10000 ug/plate
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: not specified
- Details on test system and experimental conditions:
- In the absence of S-9 mix, 100 ul of tester strain and 50 ul of vehicle, positive control, or test article were added to 2.5 ml selective top agar. When S-9 mix was required, 0.5 ml S-9, 100 ul tester strain, and 50 ul of vehicle, positive control, or test article were added to 2.0 ml selective top agar. After votexing, the agar mixture was overlaid onto the surface of plates containing minimal bottom agar. Plates were allowed to solidify, then inverted and incubated for 48 hr at 37 degrees C. Plates not counted immediately after 48 hr were stored at 4 degrees C.
Test concentrations of propyl acetate (0, 100, 333, 1000, 3333, and 10,000 ug/plate) were prepared using dimethylsulfoxide (DMSO) as the solvent; a maximum of 0.5 ml solvent was added to each plate. Each dose was tested in triplicate without activation, and with 10% rat liver S-9. Concurrent positive and solvent controls were run with each trial. - Evaluation criteria:
- A material was considered mutagenic if it produced a doubling in the mean reverants per plate in at least one tester strain. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.
- Species / strain:
- other: S. typhimurium TA100, TA 98, TA1535, TA1537, and TA1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- The test substance was non-mutagenic in the Salmonella typhimurium plate incorporation assay.
- Executive summary:
The bacterival reverse mutation assay (Ames) was conducted to investigate the genotoxic potential of the test substance. Ames was performed according former OECD guideline 471 and did not include E.Coli strain or TA 102. Five strains of S. typhimurium (TA 100, TA 98, TA 1535, TA 1537, and TA 1538) were treated with 100, 333, 1000, 3333, or 10000 ug/plate both with and without a metabolic activation system (Arochlor 1254-induced rat liver S9-mix). Adequate controls were included. No evidence of a genotoxic potential was observed for any strain tested. Cytotoxicity was not reported but the test substance was tested up to limit concentrations. As a result, the test substance was non-mutagenic in the Salmonella typhimurium plate incorporation assay.
Referenceopen allclose all
Table 1: Analysis of micronuclei - 1st Experiment;
4 hours exposure, 24 hours harvest time, without S9 mix
Test groups |
Culture |
No. of Cells |
Cells containing Micronuclei |
||
|
n |
n |
% |
||
Negative control* |
A |
1000 |
9 |
0.9 |
|
|
|
B |
1000 |
12 |
1.2 |
Test substance |
|
Total |
2000 |
21 |
1.1 |
275.0 |
µg/mL |
A |
1000 |
11 |
1.1 |
|
|
B |
1000 |
11 |
1.1 |
|
|
Total |
2000 |
22 |
1.1 |
550.0 |
µg/mL |
A |
1000 |
12 |
1.2 |
|
|
B |
1000 |
11 |
1.1 |
|
|
Total |
2000 |
23 |
1.2 |
1100.0 |
µg/mL |
A |
1000 |
11 |
1.1 |
|
|
B |
1000 |
6 |
0.6 |
|
|
Total |
2000 |
17 |
0.9 |
MMC 0.125 |
µg/mLS |
A |
1000 |
49 |
4.9 |
|
|
B |
1000 |
29 |
2.9 |
|
|
Total |
2000 |
78 |
3.9 |
* RPMI 1640
SFrequency statistically significant higher than corresponding control values
Table 2: Analysis of micronuclei – 2nd Experiment;
4 hours exposure, 24 hours harvest time, with S9 mix
Test groups |
Culture |
No. of Cells |
Cells containing Micronuclei |
||
|
n |
n |
% |
||
|
A |
1000 |
13 |
1.3 |
|
Negative control* |
B |
1000 |
9 |
0.9 |
|
Test substance |
|
Total |
2000 |
22 |
1.1 |
275.00 |
µg/mL |
A |
1000 |
12 |
1.2 |
|
|
B |
1000 |
9 |
0.9 |
|
|
Total |
2000 |
21 |
1.1 |
550.00 |
µg/mL |
A |
1000 |
10 |
1.0 |
|
|
B |
1000 |
9 |
0.9 |
|
|
Total |
2000 |
19 |
1.0 |
1100.00 |
µg/mL |
A |
1000 |
9 |
0.9 |
|
|
B |
1000 |
12 |
1.2 |
|
|
Total |
2000 |
21 |
1.1 |
CPP 0.5 |
µg/mL |
A |
1000 |
21 |
2.1 |
|
|
B |
1000 |
12 |
1.2 |
|
|
Total |
2000 |
33 |
1.7 |
CPP 1.0 |
µg/mLS |
A |
1000 |
38 |
3.8 |
|
|
B |
1000 |
41 |
4.1 |
|
|
Total |
2000 |
79 |
4.0 |
* RPMI 1640
SFrequency statistically significant higher than corresponding control values
Table 3: Analysis of micronuclei – 3rd Experiment;
24 hours exposure, 24 hours harvest time, without S9 mix
Test groups |
Culture |
No. of Cells |
Cells containing Micronuclei |
||
|
n |
n |
% |
||
Negative control* |
A |
1000 |
10 |
1.0 |
|
|
|
B |
1000 |
7 |
0.7 |
Test substance |
|
Total |
2000 |
17 |
0.9 |
550.0 |
µg/mL |
A |
1000 |
10 |
1.0 |
|
|
B |
1000 |
8 |
0.8 |
|
|
Total |
2000 |
18 |
0.9 |
800.0 |
µg/mL |
A |
1000 |
7 |
0.7 |
|
|
B |
1000 |
8 |
0.8 |
|
|
Total |
2000 |
15 |
0.8 |
1100.0 |
µg/mL |
A |
1000 |
5 |
0.5 |
|
|
B |
1000 |
7 |
0.7 |
|
|
Total |
2000 |
12 |
0.6 |
MMC 0.015 |
µg/mL |
A |
1000 |
11 |
1.1 |
|
|
B |
1000 |
13 |
1.3 |
|
|
Total |
2000 |
24 |
1.2 |
MMC 0.030 |
µg/mLS |
A |
1000 |
21 |
2.1 |
|
|
B |
1000 |
22 |
2.2 |
|
|
Total |
2000 |
43 |
2.2 |
* RPMI 1640
SFrequency statistically significant higher than corresponding control values
Table 4: Analysis of micronuclei – 3rd Experiment;
4 hours exposure, 44 hours harvest time, with S9 mix
Test groups |
Culture |
No. of Cells n |
Cells containing Micronuclei n % |
|
Negative control*
Test substance 550.0 µg/mL
800.0 µg/mL
1100.0 µg/mL
CPP 0.5 µg/mL
CPP 1.0 µg/mLS |
A |
1000 |
8 |
0.8 |
B |
1000 |
12 |
1.2 |
|
Total |
2000 |
20 |
1.0 |
|
A |
1000 |
9 |
0.9 |
|
B |
1000 |
24 |
2.4 |
|
Total |
2000 |
33 |
1.7 |
|
A |
1000 |
8 |
0.8 |
|
B |
1000 |
7 |
0.7 |
|
Total |
2000 |
15 |
0.8 |
|
A |
1000 |
11 |
1.1 |
|
B |
1000 |
16 |
1.6 |
|
Total |
2000 |
27 |
1.4 |
|
A |
1000 |
11 |
1.1 |
|
B |
1000 |
13 |
1.3 |
|
Total |
2000 |
24 |
1.2 |
|
A |
1000 |
59 |
5.9 |
|
B |
1000 |
42 |
4.2 |
|
Total |
2000 |
101 |
5.1 |
* RPMI 1640
SFrequency statistically significant higher than corresponding control values
Table 1: Mutant frequency and viability - 1stExperiment without S9 mix; 4-hour exposure period
Test groups [µg/mL] |
Cloning efficiency 2 (viability) |
Mutant frequency |
||||||
Number of colonies |
CE2 |
Number of coloniesa |
MF (per 106cells) |
|||||
Dish 1 |
Dish 2 |
Abs. [%] |
Rel. [%] |
Flask 1 |
Flask 2 |
Uncorrected |
Correctedb |
|
Negative control |
115 |
146 |
65.3 |
100.0 |
2 |
1 |
0.75 |
1.15 |
68.8 |
n.c.1 |
n.c.1 |
||||||
137.5 |
120 |
150 |
67.5 |
103.4 |
5 |
7 |
3.00 |
4.44 |
275.0 |
164 |
154 |
79.5 |
121.8 |
0 |
0 |
0.00 |
0.00 |
550.0 |
154 |
149 |
75.8 |
116.1 |
7 |
5 |
3.00 |
3.96 |
1100.0 |
154 |
172 |
81.5 |
124.9 |
1 |
0 |
0.25 |
0.31 |
EMS 400.0 |
141 |
127 |
67.0 |
102.7 |
245 |
254 |
124.75 |
186.19S |
SMutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)
Table 2: Mutant frequency and viability - 1st Experiment with S9 mix; 4-hour exposure period
Test groups [µg/mL] |
Cloning efficiency 2 (viability) |
Mutant frequency |
||||||
Number of colonies |
CE2 |
Number of coloniesa |
MF (per 106cells) |
|||||
Dish 1 |
Dish 2 |
Abs. [%] |
Rel. [%] |
Flask 1 |
Flask 2 |
Uncorrected |
Correctedb |
|
Negative control |
113 |
114 |
56.8 |
100.0 |
4 |
5 |
2.25 |
3.96 |
68.8 |
n.c.1 |
n.c.1 |
||||||
137.5 |
114 |
129 |
60.8 |
107.0 |
7 |
8 |
3.75 |
6.17 |
275.0 |
126 |
105 |
57.8 |
101.8 |
1 |
3 |
1.00 |
1.73 |
550.0 |
100 |
102 |
50.5 |
89.0 |
0 |
5 |
1.25 |
2.48 |
1100.0 |
159 |
121 |
70.0 |
123.3 |
3 |
4 |
1.75 |
2.50 |
DMBA 1.25 |
75 |
91 |
41.5 |
73.1 |
120 |
117 |
59.25 |
142.77S |
S Mutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)
Table 3: Mutant frequency and viability – 3rdExperiment without S9 mix; 4-hour exposure period
Test groups [µg/mL] |
Cloning efficiency 2 (viability) |
Mutant frequency |
||||||
Number of colonies |
CE2 |
Number of coloniesa |
MF (per 106cells) |
|||||
Dish 1 |
Dish 2 |
Abs. [%] |
Rel. [%] |
Flask 1 |
Flask 2 |
Uncorrected |
Correctedb |
|
Negative control |
163 |
154 |
79.3 |
100.0 |
3 |
8 |
2.75 |
3.47 |
87.5 |
n.c.1 |
n.c.1 |
||||||
175.0 |
151 |
135 |
71.5 |
90.2 |
8 |
11 |
4.75 |
6.64 |
350.0 |
131 |
137 |
67.0 |
84.5 |
7 |
4 |
2.75 |
4.10 |
700.0 |
127 |
152 |
69.8 |
88.0 |
7 |
3 |
2.50 |
3.58 |
1100.0 |
135 |
133 |
67.0 |
84.5 |
5 |
4 |
2.25 |
3.36 |
EMS 400.0 |
94 |
98 |
48.0 |
60.6 |
136 |
182 |
79.50 |
165.63S |
SMutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)
Table 4: Mutant frequency and viability – 4thExperiment with S9 mix; 4-hour exposure period
Test groups [µg/mL] |
Cloning efficiency 2 (viability) |
Mutant frequency |
||||||
Number of colonies |
CE2 |
Number of coloniesa |
MF (per 106cells) |
|||||
Dish 1 |
Dish 2 |
Abs. [%] |
Rel. [%] |
Flask 1 |
Flask 2 |
Uncorrected |
Correctedb |
|
Negative control |
134 |
154 |
72.0 |
100.0 |
3 |
3 |
1.50 |
2.08 |
87.5 |
n.c.1 |
n.c.1 |
||||||
175.0 |
165 |
149 |
78.5 |
109.0 |
1 |
4 |
1.25 |
1.59 |
350.0 |
145 |
179 |
81.0 |
112.5 |
3 |
3 |
1.50 |
1.85 |
700.0 |
147 |
165 |
78.0 |
108.3 |
0 |
1 |
0.25 |
0.32 |
1100.0 |
134 |
151 |
71.3 |
99.0 |
5 |
7 |
3.00 |
4.21 |
DMBA 1.25 |
118 |
91 |
52.3 |
72.6 |
227 |
211 |
109.50 |
209.57S |
SMutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro
Bacteria
Propyl acetate was negative in a guideline conform Ames test (Microbiological Associates 1989 and OECD SIDS).
The bacterival reverse mutation assay (Ames) was conducted to investigate the genotoxic potential of the test substance. Ames was performed according former OECD guideline 472 and did not include E.Coli strain or TA 102. Five strains of S. typhimurium (TA 100, TA 98, TA 1535, TA 1537, and TA 1538) were treated with 100, 333, 1000, 3333, or 10000 ug/plate both with and without a metabolic activation system (Arochlor 1254-induced rat liver S9-mix). Adequate controls were included. No evidence of a genotoxic potential was observed for any strain tested. Cytotoxicity was not reported but the test substance was tested up to limit concentrations. As a result, the test substance was non-mutagenic in the Salmonella typhimurium plate incorporation assay.
Chromosome aberration
The test substance was assessed for its potential to induce micronuclei in TK6 cells in vitro (clastogenic or aneugenic activity). Three independent experiments were carried out 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. The test groups printed in bold type were evaluated.
1st Experiment
4 hours exposure, 24 hours harvest time, without S9 mix
0; 34.38; 68.75; 137.50;275.00; 550.00and1100.00 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix (not valid)
0; 34.38; 68.75; 137.50;275.00; 550.00and1100.00 μg/mL
2nd Experiment
4 hours exposure, 24 hours harvest time, with S9 mix
0; 137.50;275.00; 550.00and1100.00 μg/mL
3rd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix
0; 200.00; 400.00;550.00; 800.00and1100.00 μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix
0; 200.00; 400.00;550.00; 800.00and1100.00 μg/mL
In the 1st Experiment cultures treated with the positive control in the presence of S9 mix did not show an increase in the micronucleus frequency. Thus, this experimental part is considered as invalid and was repeated in the 2nd Experiment. The results of the invalid part of the 1st Experiment will not be shown in this report. The raw data of this experimental part will be archived with the raw data of this study. A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.
The negative controls gave frequencies of micronucleated cells within our historical negative control data range for TK6 cells. Both positive control substances, mitomycin C (MMC) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei.
Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in human TK6 cells in the absence and the presence of metabolic activation.
On the basis of the results of the present study, the test substance did not cause any
biologically relevant increase in the number of cells containing micronuclei either without S9
mix or after adding a metabolizing system.
Gene mutation
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. Four independent experiments were carried out, with and/or 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 top concentration was determined to be the limit concentration of approx. 10 mM (1100 μg/mL). In the main experiments the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:
1st Experiment
without S9 mix
0; 68.8;137.5; 275.0; 550.0; 1100.0 μg/mL
with S9 mix
0; 68.8;137.5; 275.0; 550.0; 1100.0 μg/mL
2nd Experiment (not valid; data not shown)
without S9 mix
0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL
with S9 mix
0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL
3rd Experiment
without S9 mix
0; 87.5;175.0; 350.0; 700.0; 1100.0 μg/mL
with S9 mix (not valid; data not shown)
0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL
4th Experiment
with S9 mix
0; 87.5;175.0; 350.0; 700.0; 1100.0 μg/mL
Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week.
Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The negative controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.
In this study, in all experiments, in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.
Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in all experiments performed independently of each other.
Mitotic aneuploidy assays in the absence of metabolic activation with the yeast, Saccharomyces cerevisiae, provided negative and ambiguous results for propyl acetate (Zimmermann et al. 1985, Zimmermann et al. 1988, Zimmermann et al. 1989; reliability score 3).
Analogous substances:
Butyl acetate
Butyl acetate (maximum concentration: 2 mg/mL) was negative for chromosomal aberrations in an in vitro test using Chinese hamster lung cells without metabolic activation (Ishidate et al. 1984).
Propan-1-ol
In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA 1535, TA 100, TA 1537, TA 98) and E.coli (E. coli WP2 uvrA) were exposed to propan-1 -ol using the standard plate test (SPT) and the preincubation test (PIT) in the presence and the absence of mammalian metabolic activation. The tests were performed using five different concentrations up to 5000µg/plate. There was no evidence of induced mutant colonies over background. The study was performed under GLP conditions and satisfies the requirements of the OECD guideline 471 (BASF SE, 2009).
In an OECD guideline conform in vitro mammalian cell gene mutation assay (HPRT test, OECD 476) Chinese hamster ovary (CHO) cells were exposed to propan-1-ol at 4 different concentrations of up to 600 µg/ml (10 mM) in the presence and absence of mammalian metabolic activation. Under the experimental conditions of this study, the test item propan-1-ol was not mutagenic in the HPRT locus assay (BASF SE, 2010).
In a mammalian cell cytogenetics assay (chromosome aberration), V79 cultures were exposed to propan-1-ol (99.8% pure, vehicle: Minimum essential medium) at concentrations between 75 and 600 µg/ml (with 2 fold increment in the concentration) with and without S9 mix. The cells were exposed for 4h and 18 hours. Harvesting was performed for each time point 18 and 28 hours after the start of incubation. Tested up to the limit concentration of 10 mM (600 µg/ml), no cytotoxicity was observed in V79 cells. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background (BASF AG, 2003). The study was performed according GLP standards and also satisfies the requirement for Test Guideline (in vitro mammalian cytogenetics (chromosome aberration) OECD 473 (chinese hamster lungs fibroblast, V79).
Propan-1 -ol (maximum dose: 6000 µg/mL) was negative in vitro SCE assays conducted in Chinese hamster lung cells with and without metabolic activation (Van der Hude et al. 1987).
In vivo
no data available
Read across justification to propan-1-ol and n-butyl acetate for filling data gaps of n-propyl acetate:
As indicated by toxicokinetic studies (see chapter on toxicokinetics, metabolism and distribution), n-propyl acetate is rapidly hydrolyzed to propan-1-ol and acetate (acetic acid). Available data on propan-1-ol is therefore suitable for filling data gaps of n-propyl acetate. N-propyl acetate and n-butyl acetate differ structurally by only one –CH2 group and both substances have a similar toxicological profile. The available data for n-butyl acetate is therefore suitable for filling the data gaps of n-propyl acetate due to structural similarities. For a detailed justification of read-across, please refer to IUCLID section 13.
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008
The available experimental test data are reliable and suitable for the purpose of classification under Regulation 1272/2008. Based on the criteria laid down in Regulation (EC) No. 1272/2008, as amended for the second time in Directive EC 286/2011, classification as a mutagen is not warranted.
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.