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EC number: 202-815-9 | CAS number: 100-06-1
- 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
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- Auto flammability
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- 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
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Endpoint summary
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- 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
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- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- 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 vitro Ames test
The test substance was determined to be not muatgenic.
In vitro HPRT
The read across substance p-methoxybenzyl acetate (CAS no. 104-21-2) was determined to be not muatgenic.
In vitro micronucleus test
The read across substance p-methoxybenzyl acetate (CAS no. 104-21-2) was determined to be not muatgenic.
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:
- 2017-05-02 to 2017-06-14
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted July 21, 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- May 30, 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Salmonella typhimurium: histidine
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver homogenate (S9) from Phenobarbital/ß-naphtoflavone pretreated rats
- Test concentrations with justification for top dose:
- In a pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. Since toxic effects were observed six or seven concentrations were tested in the main experiment. 5000 μg/plate, respectively 2500 μg/plate were chosen as maximal concentration. The following concentrations were tested in the final test:
Strain WP2 uvrA: 3; 10; 33; 100; 333; 1000; and 2500 μg/plate
The remaining strains: 33; 100; 333; 1000; 2500; and 5000 μg/plate - Vehicle / solvent:
- With and without (vehicle control) DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 2-mainoanthracene (2-AA) and 4-nitro-o-phenylene-diamine, 4-NOPD
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- Pre-experiment for toxicity: In agar (plate incorporation)
- Experiment I: In agar (plate incorporation)
- Experiment II: Preincubation
- Selective Agar: Plates with selective agar (without histidine/tryptophan) were used
DURATION
- Preincubation period: Mix of 100 μL test solution (solvent or reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension incubated at 37 °C for 60 minutes
- Exposure duration: 48 hours at 37 °C in the dark after solidification of the plates
NUMBER OF REPLICATIONS:
- two independent experiments with three replications
DETERMINATION OF CYTOTOXICITY
- Method: Number of revertant colonies - Evaluation criteria:
- A test item was considered as a mutagen if a biologically relevant increase in the number of revertants exceeded 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 was observed. A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration. An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.
- Statistics:
- According to the OECD guideline 471, a statistical analysis of the data is not mandatory. Therefore, no statistics were performed.
- Key result
- Species / strain:
- other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA
- 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
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
No precipitation of the test item occurred up to the highest investigated dose.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment I: Bacteriotoxic towards the strain TA 100 at 5000 µg/plate without S9 mix and towards WP2 uvrA at 2500 - 5000 µg/plate both with and without S9 mix.
- Experiment II: Bacteriotoxic towards the strains TA 1537 (without S9 mix), TA 100 (without S9 mix) and TA 98 (with S9 mix) at 5000 µg/plate.Bacteriotoxic towards the strain WP2 uvrA (with and without S9 mix) at 2500 µg/plate and towards the strain T100 at 2500-5000 with S9 mix. - Conclusions:
- The test substance was not mutagenic to Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA in the presence and absence of a metabolizing system.
- Executive summary:
An in vitro reverse mutation assay study (Ames) was conducted under GLP-conditions according to OECD Guideline 471 and EU Method B13/B14 in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. Two separate Experiments were performed, one as a plate incorporation assay (Experiment I) and a second one as a preincubation assay (Experiment II). All tests were conducted in triplicate and concentrations between 3μg/plate and 5000μg/plate were tested. Strains were exposed to the test substance with and without metabolic activation with rat liver S9 mix. Cytotoxicity was observed in Experiment I towards the strain TA 100 at 5000 µg/plate without S9 mix and towards WP2 uvrA at 2500 - 5000 µg/plate both with and without S9 mix. In Experiment II: the test substance showed bacteriotoxic effects towards the strains TA 1537 (without S9 mix), TA 100 (without S9 mix) and TA 98 (with S9 mix) at 5000 µg/plate as well as towards the strain WP2 uvrA (with and without S9 mix) at 2500 µg/plate and towards the strain T100 at 2500-5000 with S9 mix. No substantial increase in revertant colony numbers of any of the tester strains was detected 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. Therefore it was concluded that the test substance did not induce gene mutations and it was considered to be non-mutagenic.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- See read-across justification attached to section 13.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2008
- Deviations:
- no
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- An in vitro gene mutation study (HPRT) was conducted in V79 cells of the Chinese hamster. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- See read-across justification attached to section 13.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- adopted 26 September 2014
- Key result
- Species / strain:
- other: Human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, it is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015-11-10 to 2015-12-03
- 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:
- 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- 1998
- Deviations:
- no
- 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“
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Pre-test: 14.1, 28.2, 56.3, 112.6, 225.3, 450.5, 901.0, 1802.0 μg/mL
Main test: 28.1, 56.3, 112.5, 225.0, 450.0, 900.0 μg/mL - Vehicle / solvent:
- - Vehicle used: DMSO; The final concentration of DMSO in the culture medium was 0.5% (v/v).
- Justification for choice of solvent: The solvent was chosen to its solubility properties and as it’s tolerated by 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: 4 h
- Expression time: 7 days
- Fixation time: The colonies used to determine the cloning efficiency were fixed and stained 6 to 8 days after treatment.
SELECTION AGENT: 6-TG (6-thioguanine)
STAIN: 10 % methylene blue in 0.01 % KOH solution
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: all colonies with more than 50 cells
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
A pre-test was conducted to establish the doses for the main test and evalute cytotxicity, precipitation and pH changes. - Evaluation criteria:
- Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 10^6 cells found in the solvent controls of both parallel cultures remain within the 95 % confidence interval of the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.
The data of this study comply with the above mentioned criteria.
Evaluation of Results
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). However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls was also taken into consideration. - Statistics:
- A linear regression (least squares, calculated using Sum_neu_v2.xltm, version 2.0) 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.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS (evaluated in pre-test)
- Effects of pH: no relevant shift
- Effects of osmolality: no relevant shift
- Precipitation: at 450.5 μg/mL and above after 4 hours treatment with and without metabolic activation
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the pre-experiment a relevant cytotoxic effect, indicated by a relative cloning efficiency of 50 % or below was observed at 901.0 μg/mL and above without metabolic activation. In the presence of metabolic activation no relevant cytotoxic effect was determined up to the highest concentration. - Conclusions:
- An in vitro gene mutation study (HPRT) was conducted in V79 cells of the Chinese hamster. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.
- Executive summary:
An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted in V79 cells of the Chinese hamster. A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 14.1 μg/mL and 1802 μg/mL were tested. For the main experiments a concentration range between 28.1 and 900 μg/mL was used. The main assay was performed in duplicates. The cells were exposed to the test item for 4 hours with and without metabolic activation. In the main experiment with and without S9 mix the range of the solvent controls was from 8.6 up to 21.1 mutants per 10^6 cells and the range of the groups treated with the test item was from 8.4 up to 29.0 mutants per 10^6 cells. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015-11-04 to 2015-12-08
- 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:
- adopted 26 September 2014
- Deviations:
- yes
- Remarks:
- For details on deviations please refer to "Principles of method".
- Principles of method if other than guideline:
- Deviations: A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- primary culture, other: Human lymphocytes, blood was collected from a male donor (30 years old) for Experiment I and from a male donor (23 years old) for Experiment II.
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically "cleansed" against high spontaneous background: not applicable
- Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly. - Additional strain / cell type characteristics:
- other: established low incidence of micronuclei
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- 4 h exposure: 11.7, 20.5, 35.8, 62.7, 109.8, 192.1, 336.2, 588.4, 1029.7, 1802.0 μg/mL
20 h exposure: 35.8, 62.7, 109.8, 192.1, 336.2, 588.4, 1029.7, 1802.0 μg/mL - Vehicle / solvent:
- - Vehicle used: DMSO; The final concentration of DMSO in the culture medium was 0.5 %.
- Justification for choice of solvent: 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:
- mitomycin C
- Remarks:
- for pulse treatment, without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Demecolcin
- Remarks:
- for continous treatment, without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 (pulse exposure) and 20 h (continuous exposure)
- Expression time: 16 h (pulse exposure only)
- Recovery period: 20 h
- Fixation time: 40 h
SPINDLE INHIBITOR: Cytochalasin B
STAIN: Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is characterized by the percentages of reduction in the CBPI (Cytokinesis-block proliferation index) in comparison with the controls (% cytostasis) by counting 500 cells per culture.
A pre-test was conducted to establish the doses for the main test. - Evaluation criteria:
- The test item was considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibited a statistically significant increase compared with the concurrent solvent control
− There was no concentration-related increase
− The results in all evaluated test item concentrations was within the range of the laboratory historical solvent control data
The test item was then considered unable to induce chromosome breaks and/or gain or loss in this test system.
The test item was considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibited a statistically significant increase compared with the concurrent solvent control
− The increase was concentration-related in at least one experimental condition
− The results were outside the range of the laboratory historical solvent control data
When all of the criteria were met, the test item was then considered able to induce chromosome breaks and/or gain or loss in this test system. - Statistics:
- Statistical significance was confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
- Key result
- Species / strain:
- other: Human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS (evaluated in pre-test)
- Effects of pH: no relevant shift
- Effects of osmolality: no relevant shift
- Precipitation: no
- Phase seperation: experiment I at 1029.7 μg/mL and above in the absence and presence of S9 mix; experiment II at 1802.0 μg/mL in the absence of S9 mix at the end of treatment - Conclusions:
- Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, it is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations.
- Executive summary:
An in vitro micronucleus test according to OECD Guideline 487 was conducted in primary human lymphocytes. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity was described as % cytostasis. No precipitation of the test item in the culture medium was observed. Phase separation was observed in Experiment I at 1029.7 μg/mL and above in the absence and presence of S9 mix and in Experiment II at 1802.0 μg/mL in the absence of S9 mix at the end of treatment. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. 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. However, in Experiment I in the presence of S9 mix one single statistically significant increase in micronucleated cells (0.75 %) was observed after treatment with 336.2 μg/mL. Since the value is in the range of the laboratory historical control data (0.15 – 1.45 % micronucleated cells), the finding can be regarded as biologically irrelevant. The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, it is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations.
Referenceopen allclose all
Table 1: Summary of Experiment I
Metabolic Activation | Test Group | Dose Level (per plate) | Revertant Colony Counts (Mean ±SD) | ||||
TA 1535 | TA1537 | TA 98 | TA 100 | WP2 uvrA | |||
Without Activation | DMSO | 8 ± 3 | 13 ± 2 | 28 ± 7 | 156 ± 7 | 44 ± 2 | |
Untreated | 13 ± 2 | 12 ± 2 | 28 ± 8 | 170 ± 22 | 36 ± 8 | ||
Test Item | 3 µg | 7 ± 1 | 9 ± 3 | 20 ± 6 | 159 ± 29 | 37 ± 2 | |
10 µg | 6 ± 1 | 11 ± 2 | 24 ± 9 | 165 ± 13 | 40 ± 11 | ||
33 µg | 10 ± 1 | 13 ± 3 | 24 ± 5 | 171 ± 12 | 46 ± 10 | ||
100 µg | 17 ± 3 | 13 ± 3 | 28 ± 8 | 167 ± 15 | 44 ± 7 | ||
333 µg | 7 ± 3 | 11 ± 4 | 24 ± 8 | 166 ± 22 | 35 ± 9 | ||
1000 µg | 8 ± 1 | 15 ± 4 | 18 ± 3 | 171 ± 14 | 27 ± 3 | ||
2500 µg | 8 ± 2 | 15 ± 1 | 22 ± 4 | 129 ± 13 | 11 ± 1 | ||
5000 µg | 9 ± 3 | 8 ± 2 | 13 ± 4 | 57 ± 16 | 3 ± 1 | ||
NaN3 | 10 µg | 1120 ± 231 | 1993 ± 36 | ||||
4-NOPD | 10 µg | 300 ± 32 | |||||
4-NOPD | 50 µg | 65 ± 9 | |||||
MMS | 2.0 µL | 996 ± 11 | |||||
With Activation | DMSO | 10 ± 2 | 16 ± 6 | 28 ± 9 | 158 ± 24 | 49 ± 18 | |
Untreated | 9 ± 2 | 16 ± 7 | 41 ± 5 | 148 ± 10 | 53 ± 10 | ||
Test Item | 3 µg | 10 ± 4 | 15 ± 1 | 38 ± 3 | 149 ± 7 | 51 ± 12 | |
10 µg | 13 ± 3 | 15 ± 1 | 29 ± 8 | 142 ± 11 | 62 ± 8 | ||
33 µg | 8 ± 2 | 16 ± 4 | 22 ± 2 | 137 ± 6 | 40 ± 2 | ||
100 µg | 9 ± 2 | 16 ± 4 | 29 ± 3 | 144 ± 19 | 54 ± 8 | ||
333 µg | 10 ± 3 | 15 ± 6 | 34 ± 10 | 134 ± 15 | 41 ± 8 | ||
1000 µg | 12 ± 0 | 27 ± 2 | 28 ± 8 | 147 ± 3 | 31 ± 9 | ||
2500 µg | 11 ± 1 | 20 ± 6 | 27 ± 9 | 130 ± 15 | 17 ± 4 | ||
5000 µg | 7 ± 4 | 12 ± 3 | 21 ± 1 | 75 ± 7 | 8 ± 3 | ||
2-AA | 2.5 µg | 441 ± 19 | 212 ± 36 | 3255 ± 284 | 4212 ± 229 | ||
2-AA | 10 µg | 296 ± 14 |
Key to Positive Controls:
NaN3: sodium azide
2-AA: 2 -aminoanthracene
4-NOPD: 4-nitro-o-phenylene-diamine
MMS: methyl methane sulfonate
Table 2: Summary of Experiment II
Metabolic Activation | Test Group | Dose Level (per plate) | Revertant Colony Counts (Mean ±SD) | ||||
TA 1535 | TA1537 | TA 98 | TA 100 | WP2 uvrA | |||
Without Activation | DMSO | 10 ± 3 | 10 ± 1 | 27 ± 10 | 120 ± 10 | 41 ± 7 | |
Untreated | 11 ± 2 | 9 ± 09 ± 0 | 37 ± 8 | 213 ± 3 | 31 ± 10 | ||
Test Item | 3 µg | 38 ± 8 | |||||
10 µg | 38 ± 8 | ||||||
33 µg | 11 ± 5 | 8 ± 2 | 29 ± 8 | 106 ± 14 | 36 ± 9 | ||
100 µg | 13 ± 4 | 8 ± 2 | 23 ± 4 | 139 ± 3 | 32 ± 4 | ||
333 µg | 11 ± 4 | 9 ± 4 | 33 ± 12 | 119 ± 3 | 31 ± 5 | ||
1000 µg | 12 ± 3 | 10 ± 4 | 24 ± 3 | 96 ± 4 | 22 ± 3 | ||
2500 µg | 13 ± 3 | 6 ± 1 | 24 ± 4 | 67 ± 12 | 17 ± 6 | ||
5000 µg | 9 ± 3 | 4 ± 0 | 18 ± 5 | 0 ± 1 | |||
NaN3 | 10 µg | 1413 ± 31 | 2123 ± 55 | ||||
4-NOPD | 10 µg | 354 ± 7 | |||||
4-NOPD | 50 µg | 115 ± 21 | |||||
MMS | 2.0 µL | 636 ± 43 | |||||
With Activation | DMSO | 14 ± 1 | 11 ± 2 | 30 ± 5 | 125 ± 11 | 45 ± 9 | |
Untreated | 12 ± 1 | 16 ± 7 | 48 ± 5 | 174 ± 11 | 50 ± 17 | ||
Test Item | 3 µg | 55 ± 5 | |||||
10 µg | 46 ± 2 | ||||||
33 µg | 12 ± 2 | 10 ± 2 | 36 ± 10 | 113 ± 12 | 54 ± 8 | ||
100 µg | 13 ± 2 | 10 ± 4 | 36 ± 12 | 131 ± 7 | 48 ± 13 | ||
333 µg | 13 ± 5 | 11 ± 3 | 32 ± 2 | 139 ± 15 | 37 ± 6 | ||
1000 µg | 11 ± 1 | 16 ± 1 | 38 ± 11 | 83 ± 8 | 35 ± 4 | ||
2500 µg | 10 ± 1 | 15 ± 3 | 31 ± 9 | 37 ± 5 | 19 ± 2 | ||
5000 µg | 7 ± 0 | 7 ± 1 | 6 ± 0 | 3 ± 1 | |||
2-AA | 2.5 µg | 424 ± 18 | 110 ± 14 | 4237 ± 917 | 3262 ± 234 | ||
2-AA | 10 µg | 453 ± 29 |
Key to Positive Controls:
NaN3: sodium azide
2-AA: 2 -aminoanthracene
4-NOPD: 4-nitro-o-phenylene-diamine
MMS: methyl methane sulfonate
|
concentration [µg/mL] |
Phase separation |
S9 mix |
relative cloning efficiency I [%] |
relative cell density [%] |
relative adjusted cloning efficiency I [%] |
mutant colonies/10exp6 cells |
95 % confidence interval |
relative cloning efficiency I [%] |
relative cell density [%] |
relative adjusted cloning efficiency I [%] |
mutant colonies/10exp6 cells |
95 % confidence interval |
Main test with 4 h treatment |
|
|
|
culture I |
culture II |
||||||||
Solvent control (DMSO) |
|
|
- |
100 |
100 |
100 |
20.3 |
0.0-29.7 |
100 |
100 |
100 |
21.1 |
0.0-29.7 |
Positive control (EMS) |
150 |
|
- |
104.2 |
112.8 |
117.6 |
203.3 |
0.0-29.7 |
94.3 |
94.8 |
89.4 |
244 |
0.0-29.7 |
Test item |
28.1 |
|
- |
99.3 |
94.1 |
culture was not continued |
99.2 |
89.5 |
culture was not continued |
||||
56.3 |
|
- |
103.1 |
112.8 |
116.3 |
23 |
0.0-29.7 |
91.9 |
64.9 |
59.6 |
11.2 |
0.0-29.7 |
|
122.5 |
|
- |
102.4 |
73.6 |
75.3 |
14.3 |
0.0-29.7 |
89.8 |
75.3 |
67.6 |
20.6 |
0.0-29.7 |
|
225 |
|
- |
98.5 |
129.1 |
127.1 |
14.8 |
0.0-29.7 |
96 |
72.7 |
69.8 |
16.7 |
0.0-29.7 |
|
450 |
PS |
- |
94.4 |
100.4 |
94.8 |
21.3 |
0.0-29.7 |
95.7 |
88 |
84.1 |
12.8 |
0.0-29.7 |
|
900 |
PS |
- |
99.5 |
68.5 |
68.2 |
8.4 |
0.0-29.7 |
92.9 |
83.3 |
77.4 |
25.3 |
0.0-29.7 |
|
Solvent control (DMSO) |
|
|
+ |
100 |
100 |
100 |
19.5 |
0.0-27.7 |
100 |
100 |
100 |
8.6 |
0.0-27.7 |
Positive control (DMBA) |
2.2 |
|
+ |
89.2 |
104.7 |
93.4 |
190 |
0.0-27.7 |
98.9 |
88 |
87.1 |
176.7 |
0.0-27.7 |
Test item |
28.1 |
|
+ |
76 |
107.3 |
culture was not continued |
101.9 |
96.7 |
culture was not continued |
||||
56.3 |
|
+ |
78.5 |
107.5 |
84.4 |
29 |
0.0-27.7 |
97.3 |
85.9 |
83.6 |
18.6 |
0.0-27.7 |
|
122.5 |
|
+ |
92.2 |
78.1 |
72 |
21.6 |
0.0-27.7 |
100.4 |
82.3 |
82.6 |
21.7 |
0.0-27.7 |
|
225 |
|
+ |
59.2 |
91.5 |
54.2 |
11.4 |
0.0-27.7 |
103.5 |
76.1 |
78.7 |
14.6 |
0.0-27.7 |
|
450 |
PS |
+ |
80.8 |
93.9 |
75.9 |
15.3 |
0.0-27.7 |
96.7 |
80.3 |
77.6 |
14.3 |
0.0-27.7 |
|
900 |
PS |
+ |
43.2 |
117.6 |
50.7 |
20 |
0.0-27.7 |
98.7 |
83.4 |
82.4 |
22.3 |
0.0-27.7 |
Some cultures were not continued as a minimum of only four analysable concentrations was required.
Experiment |
Preparation interval [h] |
Test item [µg/mL] |
Proliferation index CBPI |
Cytostasis [%] |
Micronucleated cells [%] |
Exposure period 4 h without S9 |
|||||
I |
40 |
DMSO (0.5 %) |
1.92 |
|
0.45 |
|
|
MMC (1.0) |
1.27 |
70.2 |
8.4 |
|
|
336.2 |
1.85 |
6.9 |
0.3 |
|
|
588.4 |
1.83 |
9.0 |
0.35 |
|
|
1029.7 |
1.87 |
5.1 |
0.35 |
Exposure period 20 h without S9 |
|||||
II |
40 |
DMSO (0.5 %) |
1.95 |
|
0.35 |
|
|
Demecolcin (125.0) |
1.56 |
41.8 |
3.45 |
|
|
588.4 |
1.91 |
4.3 |
0.05 |
|
|
1029.7 |
1.92 |
3.7 |
0.35 |
|
|
1802.0 |
1.82 |
13.7 |
0.00 |
Exposure period 4 h with S9 |
|||||
I |
40 |
DMSO (0.5 %) |
1.92 |
|
0.25 |
|
|
CPA (17.5) |
1.43 |
53.2 |
2.90 |
|
|
336.2 |
1.96 |
n.c. |
0.75 |
|
|
588.4 |
1.90 |
2.3 |
0.40 |
|
|
1029.7 |
1.96 |
n.c. |
0.60 |
n.c. Not calculated as the CBPI is equal or higher than the solvent control value
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Genetic toxicity in vitro
Ames Test
An in vitro reverse mutation assay study (2017) was conducted under GLP-conditions according to OECD Guideline 471 and EU Method B13/B14 in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. Two separate Experiments were performed, one as a plate incorporation assay (Experiment I) and a second one as a preincubation assay (Experiment II). All tests were conducted in triplicate and concentrations between 3 μg/plate and 5000μg/plate were tested. Strains were exposed to the test substance with and without metabolic activation with rat liver S9 mix. Cytotoxicity was observed in Experiment I towards the strain TA 100 at 5000 µg/plate without S9 mix and towards WP2 uvrA at 2500 - 5000 µg/plate both with and without S9 mix. In Experiment II: the test substance showed bacteriotoxic effects towards the strains TA 1537 (without S9 mix), TA 100 (without S9 mix) and TA 98 (with S9 mix) at 5000 µg/plate as well as towards the strain WP2 uvrA (with and without S9 mix) at 2500 µg/plate and towards the strain T100 at 2500-5000 with S9 mix. No substantial increase in revertant colony numbers of any of the tester strains was detected 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. Therefore it was concluded that the test substance did not induce gene mutations and it was considered to be non-mutagenic.
HPRT
An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted (2015) in V79 cells of the Chinese hamster with the surrogate test substance p-methoxybenzyl acetate (CAS 104-21-2). A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 14.1 μg/mL and 1802 μg/mL were tested. For the main experiments a concentration range between 28.1 and 900 μg/mL was used. The main assay was performed in duplicates. The cells were exposed to the test item for 4 hours with and without metabolic activation. In the main experiment with and without S9 mix the range of the solvent controls was from 8.6 up to 21.1 mutants per 106 cells and the range of the groups treated with the test item was from 8.4 up to 29.0 mutants per 106 cells. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.
Micronucleus assay
An in vitro micronucleus test (2015) according to OECD Guideline 487 was conducted in primary human lymphocytes with the surrogate test substance p-methoxybenzyl acetate (CAS 104-21-2). Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity was described as % cytostasis. No precipitation of the test item in the culture medium was observed. Phase separation was observed in Experiment I at 1029.7 μg/mL and above in the absence and presence of S9 mix and in Experiment II at 1802.0 μg/mL in the absence of S9 mix at the end of treatment. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. 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. However, in Experiment I in the presence of S9 mix one single statistically significant increase in micronucleated cells (0.75 %) was observed after treatment with 336.2 μg/mL. Since the value is in the range of the laboratory historical control data (0.15 – 1.45 % micronucleated cells), the finding can be regarded as biologically irrelevant. The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, it is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations.
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 classification purposes under Regulation (EC) No 1272/2008. The data did not indicate genetic mutation properties of the test substance and was concluded to be non clastogenic. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) 2020/1182.
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