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EC number: 203-602-3 | CAS number: 108-64-5
- 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
All required in vitro tests showed negative results for genetic toxicity.
The test substance is negative in Bacterial Reverse Mutation Assay for S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A with and without S9 metabolic activation.
3 -methylbutyl isovalerate as a Read-across substance did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes and therefore is considered to be non-mutagenic in this in vitro micronucleus test.
3 -methylbutyl isovalerate as a Read-across substance did not induce gene mutations at the HPRT locus in V79 cells and therefore is considered to be non-mutagenic in this HPRT assay.
Therefore, using a read across approach , the target substance is considered to be not mutagenic
According to Column 2 in Annex VIII (Section 8.4), an in vivo test is not necessary given the consistently negative results in multiple in vitro studies.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14.07.2015 - 15.10.2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- 3; 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate (experiment 2); top dose chosen following the pre-experiment (experiment 1)
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. - Untreated negative controls:
- yes
- Remarks:
- Medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA with metabolic activation
- Untreated negative controls:
- yes
- Remarks:
- Medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- TA 1535; TA 100 without metabolic activation
- Untreated negative controls:
- yes
- Remarks:
- Medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylene-diamine
- Remarks:
- TA 1537, TA 98 without metabolic activation
- Untreated negative controls:
- yes
- Remarks:
- Medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- WP2 uvrA without metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in DMSO
DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: spontaneous reversion rates - Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Key result
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100 bacteria
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The test substance is negative in Bacterial Reverse Mutation Assay for S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A with and without S9 metabolic activation, hence is classified as not mutagenic.
- Executive summary:
This study was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations in both experiments: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate. No precipitation of the test item occurred up to the highest investigated dose.
The plates incubated with the test item showed reduced background growth in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains used except strains TA 1537 and WP2 uvrA. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- March - May 2016
- 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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Guidelines: “Kanpoan No. 287 -- Environment Protection Agency“ “Eisei No. 127 -- Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry“.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: gene mutation assay in mammalian cells
- Target gene:
- HPRT
- Species / strain / cell type:
- other: Chinese hamster (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Suitability of cells: Yes
- Cell cycle length, doubling time or proliferation index: doubling time 12 - 16 h in stock cultures
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. Cell culture incubation: humidified atmosphere with 1.5% CO2 at 37°C.
- Properly maintained: [yes]
- Checked for Mycoplasma contamination: [yes]
- Checked for karyotype stability: [yes)
- "Cleansed' against high spontaneous background: [yes] - Metabolic activation:
- with and without
- Metabolic activation system:
- mammalian microsomal fraction S9 mix
- Test concentrations with justification for top dose:
- Doses applied in the gene mutation assay (concentrations marked with ¹ were chosen for the mutation rate analysis):
Experiment I (without S9, 4h exposure): 1.7, 3.4 ¹, 6.8 ¹, 13.5 ¹, 27.0 ¹, 54.0 ¹, 108.0 µg/mL
Experiment II (with S9, 4h exposure): 26.9 ¹, 53.8 ¹, 107.5 ¹, 215.0 ¹, 430.0* ¹, 860.0* µg/mL
* indicates Phase separation observed
The cultures at the lowest concentration without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines. The cultures at the highest concentration without metabolic activation were not continued based on exceedingly severe cytotoxicity. The cultures at the highest concentration with metabolic activation were not continued to avoid analysis of too many insoluble concentrations. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- With metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 7d
- Selection time (if incubation with a selection agent): 8d
SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)
NUMBER OF REPLICATIONS: 5 to determine mutation (culture flasks with medium containing 6-TG per concentration)
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Stained with 10% methylene blue in 0.01% KOH solution
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: The experimental part of the main experiment without metabolic activation was prematurely terminated as exceedingly severe cytotoxicity occurred already at low concentrations. This experimental part was repeated as experiment IA in a lower concentration range. Experiment IA was again terminated due to severe cytotoxicity and repeated as experiment IB with even lower concentrations. The data of experiment IB are reported as first experiment without metabolic activation. - Evaluation criteria:
- A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares). - Statistics:
- Statistical Analysis: A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
experimental group p-value
experiment I, culture I without S9 mix, p-value: 0.492
experiment I, culture II without S9 mix, p-value: 0.025 S
experiment I, culture I with S9 mix, p-value: 0.453
experiment I, culture II with S9 mix, p-value: 0.855
S = significant trend - Key result
- Species / strain:
- other: Chinese hamster (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiment up to the maximum concentration. The 95% confidence interval was not exceeded.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the second culture without metabolic activation. This trend however, was judged as irrelevant as all of the absolute values of the mutation frequency remained within the 95% confidence interval.
In the main experiment with and without S9 mix the range of the solvent controls was from 13.0 up to 18.1 mutants per 10E6 cells; the range of the groups treated with the test item was from 9.3 up to 26.9 mutants per 10E6 cells.
EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. - Conclusions:
- The test item did not induce gene mutations at the HPRT locus in V79 cells and therefore is considered to be non-mutagenic in this HPRT assay.
- Executive summary:
The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (1723 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiment was limited by the cytotoxicity and the solubility of the test item.
The experimental part of the main experiment without metabolic activation was prematurely terminated as exceedingly severe cytotoxicity occurred already at low concentrations. This experimental part was repeated as experiment IA in a lower concentration range. Experiment IA was again terminated due to severe cytotoxicity and repeated as experiment IB with even lower concentrations. The data of experiment IB are reported as the first experiment without metabolic activation.
No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-03-02 to 2016-06-07
- 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)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes: human
- Cytokinesis block (if used):
- Cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- 3.65; 6.40; 11.2; 19.6; 34.3; 60.0; 105; 184; 322; 563 µg/L (experiment IA)
11.2, 19.6, 34.3, 60.0, 105, 184, 322, 563, 985, 1723 µg/L (experiment IB)
18.2, 27.3, 41.0, 61.5, 92.2, 138, 207, 311, 467 µg/L (experiment II); top dose chosen due to strong cytotoxic effects (low cell numbers) at higher concentrations - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. - Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- other: Demecolcin
- Remarks:
- Without metabolic activation
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 20 min
- Exposure duration: 4 hours (experiment I; with and without S9) ; 20 hours (experiment II; without S9)
- Selection time (if incubation with a selection agent): 20 hours Cytochalasin B exposure
- Fixation time (start of exposure up to fixation or harvest of cells): 40 hours after beginning of treatment
SELECTION AGENT (mutation assays): Cytochalasin B
SPINDLE INHIBITOR (cytogenetic assays): Giemsa
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide.
NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976).
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture. - Evaluation criteria:
- A test item is considered to be clearly negative if, in all of the experimental conditions examined:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response. - Statistics:
- Statistical significance was confirmed by the Chi square test (a < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- no Cytotoxicity observed in experiment I up to phase separation
- Vehicle controls validity:
- valid
- Remarks:
- DMSO
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at highest concentration moderate cytotoxicity was observed
- Vehicle controls validity:
- valid
- Remarks:
- DMSO
- Positive controls validity:
- valid
- Conclusions:
- The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes and therefore is considered to be non-mutagenic in this in vitro micronucleus test.
- Executive summary:
The test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. In each experimental group two parallel cultures were analyzed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (1723 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487.
In the experiments IA and IB in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II in the absence of S9 mix after continuous treatment moderate cytotoxicity of 46.0 % cytostasis was observed at the highest evaluable concentration. In the experiments IA and IB in the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In Experiment II in the absence of S9 mix after continuous treatment one statistically significant increase (1.45 %) in micronucleate cells was observed after treatment with 138 µg/mL. The value exceeded the 95 % control limit of the historical control data range (0.05 – 1.05% micronucleate cells). This can be declared as biologically irrelevant because the increase is not dose-related. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating or the highest evaluable concentration.
Referenceopen allclose all
Experiment 1
Without metabolic activation
|
Dose/plate |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2uvrA |
DMSO |
|
9±2 |
11±1 |
24±5 |
140±3 |
39±5 |
Untreated |
|
11 ± 2 |
8 ± 1 |
27 ± 2 |
160 ± 10 |
44 ± 5 |
Test item |
3 µg |
8 ± 2 |
11 ± 2 |
24 ± 6 |
135 ± 5 |
40 ± 3 |
|
10 µg |
11 ± 3 |
12 ± 2 |
24 ± 3 |
158 ± 11 |
39 ± 2 |
|
33 µg |
10 ± 5 |
11 ± 3 |
19 ± 5 |
139 ± 13 |
43 ± 8 |
|
100 µg |
8 ± 1 |
10 ± 2 |
18 ± 4 |
153 ± 9 |
38 ± 8 |
|
333 µg |
9 ± 3 |
9 ± 3 |
21 ± 2 |
126 ± 5 |
42 ± 7 |
|
1000 µg |
9 ± 1 |
11 ± 4 |
27 ± 6 |
127 ± 6 |
40 ± 3 |
|
2500 µg |
7 ± 2 |
5 ± 2 |
20 ± 2 |
70 ± 4 |
38 ± 11 |
|
5000 µg |
7 ± 1 |
5 ± 2 |
18 ± 4 |
46 ± 7* |
28 ± 4 |
NaN3 |
10 µg |
919 ± 66 |
|
|
2106 ± 122 |
|
4-NOPD |
10 µg |
|
|
413 ± 9 |
|
|
4-NOPD |
50 µg |
|
90 ±5 |
|
|
|
MMS |
2.0 µL |
|
|
|
|
783 ± 68 |
With metabolic activation
|
Dose/plate |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2uvrA |
DMSO |
|
10 ± 2 |
11 ± 4 |
34 ± 8 |
117 ± 10 |
46 ± 8 |
Untreated |
|
14 ± 4 |
9 ± 4 |
36 ± 2 |
144 ± 10 |
48 ± 10 |
Test item |
3 µg |
11 ± 3 |
12 ± 4 |
34 ± 5 |
115 ± 11 |
46 ± 7 |
|
10 µg |
9 ± 1 |
12 ± 5 |
35 ± 3 |
107 ± 10 |
51 ± 7 |
|
33 µg |
9 ± 1 |
12 ± 4 |
28 ± 6 |
110 ± 15 |
46 ± 6 |
|
100 µg |
9 ± 1 |
13 ± 4 |
31 ± 4 |
116 ± 15 |
45 ± 4 |
|
333 µg |
11 ± 2 |
12 ± 3 |
29 ± 3 |
101 ± 4 |
43 ± 2 |
|
1000 µg |
8 ± 2 |
11 ± 2 |
34 ± 1 |
113 ± 6 |
43 ± 4 |
|
2500 µg |
10 ± 3 |
9 ± 3 |
33 ± 2 |
86 ± 9 |
37 ± 7 |
|
5000 µg |
9 ± 1 |
9 ± 2 |
26 ± 2 |
63 ± 11 |
44 ± 1 |
2-AA |
2.5 µg |
399 ± 8 |
225 ± 34 |
4168 ± 319 |
3201 ± 160 |
|
2-AA |
10 µg |
|
|
|
|
370 ± 37 |
Experiment 2
Without metabolic activation
|
Dose/plate |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2uvrA |
DMSO |
|
11 ± 2 |
8 ± 3 |
22 ± 4 |
176 ± 13 |
34 ± 4 |
Untreated |
|
11 ± 3 |
11 ± 2 |
32 ± 14 |
189 ± 23 |
40 ± 6 |
Test item |
3 µg |
12 ± 2 |
7 ± 1 |
23 ± 4 |
159 ± 12 |
35 ± 4 |
|
10 µg |
10 ± 4 |
7 ± 1 |
22 ± 4 |
175 ± 23 |
36 ± 2 |
|
33 µg |
10 ± 4 |
6 ± 1 |
26 ± 4 |
171 ± 20 |
33 ± 4 |
|
100 µg |
10 ± 4 |
10 ± 1 |
24 ± 8 |
164 ± 6 |
34 ± 7 |
|
333 µg |
9 ± 4 |
8 ± 1 |
22 ± 3 |
131 ± 14 |
37 ± 8 |
|
1000 µg |
8 ± 2 |
7 ± 2 |
21 ± 5 |
91 ± 13 |
28 ± 4 |
|
2500 µg |
6 ± 1* |
7 ± 2 |
27 ± 1 |
72 ± 11 |
27 ± 1* |
|
5000 µg |
4 ± 2* |
7 ± 2* |
5 ± 2* |
0 ± 0 |
22 ± 2* |
NaN3 |
10 µg |
1173 ± 24 |
|
|
1895 ± 196 |
|
4-NOPD |
10 µg |
|
|
428 ± 28 |
|
|
4-NOPD |
50 µg |
|
103 ± 19 |
|
|
|
MMS |
2.0 µL |
|
|
|
|
580 ± 90 |
With metabolic activation
|
Dose/plate |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2uvrA |
DMSO |
|
9 ± 1 |
10 ± 3 |
28 ± 2 |
146 ± 14 |
43 ± 7 |
Untreated |
|
12 ± 3 |
12 ± 2 |
40 ± 9 |
212 ± 8 |
49 ± 8 |
Test item |
3 µg |
9 ± 3 |
12 ± 2 |
31 ± 3 |
143 ± 6 |
44 ± 6 |
|
10 µg |
10 ± 4 |
9 ± 5 |
33 ± 9 |
151 ± 40 |
47 ± 14 |
|
33 µg |
13 ± 1 |
9 ± 4 |
33 ± 2 |
148 ± 23 |
50 ± 11 |
|
100 µg |
9 ± 2 |
10 ± 4 |
29 ± 3 |
139 ± 8 |
52 ± 7 |
|
333 µg |
10 ± 1 |
13 ± 4 |
33 ± 5 |
136 ± 14 |
50 ± 8 |
|
1000 µg |
10 ± 4 |
8 ± 3 |
33 ± 4 |
109 ± 4 |
47 ± 12 |
|
2500 µg |
13 ± 3 |
8 ± 3 |
24 ± 4 |
57 ± 7 |
32 ± 5 |
|
5000 µg |
13 ± 1* |
7 ± 3* |
26 ± 7 |
40 ± 4 |
21 ± 2* |
2-AA |
2.5 µg |
372 ± 72 |
174 ± 11 |
3958 ± 855 |
4259 ± 350 |
|
2-AA |
10 µg |
|
|
|
|
298 ± 8 |
* Reduced background count
Summary of results
Experiment |
Preparation interval |
Test item concentration in µg/mL |
Proliferation index CBPI |
Cytostasis in %* |
Micronucleated cells in %** |
|
|
Exposure period 4 hrs without S9 mix |
|||
IB |
40 hrs |
Solvent control1 |
2.01 |
|
0.15 |
|
|
Positive control2 |
1.81 |
19.9 |
10.70S |
|
60.0 |
2.01 |
0.6 |
0.10 |
|
|
105 |
1.86 |
14.9 |
0.25 |
|
|
184PS |
1.86 |
15.3 |
0.20 |
|
|
|
Exposure period 20 hrs without S9 mix |
|||
II |
40 hrs |
Solvent control1 |
1.95 |
|
0.75 |
|
|
Positive control3 |
1.54 |
42.5 |
3.90S |
|
|
92.2 |
1.92 |
3.0 |
0.75 |
|
|
138# |
1.72 |
23.5 |
1.45S |
|
|
207 |
1.51 |
46.0 |
0.90 |
|
|
Exposure period 4 hrs with S9 mix |
|||
IA |
40 hrs |
Solvent control1 |
1.88 |
|
0.20 |
|
|
Positive control4 |
1.49 |
44.3 |
5.85S |
|
|
105 |
2.02 |
n.c. |
0.20 |
|
|
184 |
2.00 |
n.c. |
0.35 |
|
|
322PS |
1.91 |
n.c. |
0.35 |
* For the positive control groups and the test item treatment groups the values are related to the solvent controls
** The number of micronucleated cells was determined in a sample of 2000 binucleated cells
#The number of micronucleated cells was determined in a sample of 4000 binucleated cells
PS Phase separation occurred at the end of treatment
S The number of micronucleated cells is statistically significantly higher than corresponding control values
n.c. Not calculated as the CBPI is equal or higher than the solvent control value
1 DMSO 1.0 % (v/v)
2 MMC 1.0 µg/mL
3 Demecolcin 125.0 µg/mL
4 CPA 15.0 µg/mL
Toxicity – Experiment IA
Concentration (µg/mL) |
Exposure time |
Preparation interval |
CBPI per 500 cells* |
Cytostasis (%) |
|
With S9 mix |
|||
Solvent control |
4 hrs |
40 hrs |
1.88 |
|
3.65 |
4 hrs |
40 hrs |
n.d. |
n.d. |
6.40 |
4 hrs |
40 hrs |
n.d. |
n.d. |
11.2 |
4 hrs |
40 hrs |
n.d. |
n.d. |
19.6 |
4 hrs |
40 hrs |
n.d. |
n.d. |
34.3 |
4 hrs |
40 hrs |
1.91 |
n.c. |
60.0 |
4 hrs |
40 hrs |
1.98 |
n.c. |
105 |
4 hrs |
40 hrs |
2.02 |
n.c. |
184 |
4 hrs |
40 hrs |
2.00 |
n.c. |
322PS |
4 hrs |
40 hrs |
1.91 |
n.c. |
563PS |
4 hrs |
40 hrs |
1.94 |
n.c. |
985PS |
4 hrs |
40 hrs |
1.90 |
n.c. |
1723PS |
4 hrs |
40 hrs |
1.88 |
n.c |
Experimental groups evaluated for cytogenetic damage are shown in bold characters
* Mean value of two cultures in %
PS Phase separation occurred at the end of treatment
n.d. Not determined
n.c. Not calculated as the CBPI was equal or higher than solvent control value
Toxicity – Experiment IB
Concentration (µg/mL) |
Exposure time |
Preparation interval |
CBPI per 500 cells* |
Cytostasis (%) |
|
Without S9 mix |
|||
Solvent control |
4 hrs |
40 hrs |
2.01 |
|
11.2 |
4 hrs |
40 hrs |
2.05 |
n.c. |
19.6 |
4 hrs |
40 hrs |
2.03 |
n.c. |
34.3 |
4 hrs |
40 hrs |
2.08 |
n.c. |
60.0 |
4 hrs |
40 hrs |
2.01 |
0.6 |
105 |
4 hrs |
40 hrs |
1.86 |
14.9 |
184PS |
4 hrs |
40 hrs |
1.86 |
15.3 |
322PS |
4 hrs |
40 hrs |
1.61 |
39.6 |
563PS |
4 hrs |
40 hrs |
1.86 |
15.5 |
985PS |
4 hrs |
40 hrs |
1.65 |
36.1 |
1723PS |
4 hrs |
40 hrs |
n.e. |
n.e. |
Experimental groups evaluated for cytogenetic damage are shown in bold characters
* Mean value of two cultures in %
PS Phase separation occurred at the end of treatment
n.c. Not calculated as the CBPI was equal or higher than solvent control value
n.e. Not evaluable due to strong cytotoxic effects
Toxicity – Experiment II
Concentration (µg/mL) |
Exposure time |
Preparation interval |
CBPI per 500 cells* |
Cytostasis (%) |
|
Without S9 mix |
|||
Solvent control |
20 hrs |
40 hrs |
1.95 |
|
18.2 |
20 hrs |
40 hrs |
n.d. |
n.d. |
27.3 |
20 hrs |
40 hrs |
n.d. |
n.d. |
41.0 |
20 hrs |
40 hrs |
n.d. |
n.d. |
61.5 |
20 hrs |
40 hrs |
1.96 |
n.c. |
92.2 |
20 hrs |
40 hrs |
1.92 |
3.0 |
138 |
20 hrs |
40 hrs |
1.72 |
23.5 |
207 |
20 hrs |
40 hrs |
1.51 |
46.0 |
311 |
20 hrs |
40 hrs |
1.52 |
45.0 |
467PS |
20 hrs |
40 hrs |
n.e. |
n.e. |
Experimental groups evaluated for cytogenetic damage are shown in bold characters
* Mean value of two cultures in %
PS Phase separation occurred at the end of treatment
n.d. Not determined
n.c. Not calculated as the CBPI was equal or higher than solvent control value
n.e. Not evaluable due to strong cytotoxic effects
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Link to relevant study records
- Endpoint:
- genetic toxicity in vivo, other
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- other:
Reference
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Information are available from reliable studies for all the required in vitro endpoints. The results of all studies were in agreement.
Where there was more than one reliable study for an endpoint the most recent studies were selected as key studies.
In vitro key and supporting studies indicate that the test substance is negative in the gene mutation (Bacterial reverse mutation assay / Ames test) test and it is predicted from a read a cross substance 3 -methylbutyl isovalerate in the key mammalian cell gene mutation test/ HPRT, with and without metabolic activation.
One key in vitro mammalian micronucleus study for a read a cross substance 3 -methylbutyl isovalerate as well as one supporting for the test item are available for in vitro mammalian chromosome aberration and, respectively, to determine the genetic toxicity of the substance. The results show that the substance does not induce chromosome aberration or micronuclei in mammalian cells.
Justification for classification or non-classification
Based on the available data, the substance does not require classification for mutagenicity according to Regulation (EC) No 1272/2008.
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.
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