Registration Dossier
Registration Dossier
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EC number: 208-736-6 | CAS number: 540-10-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test (OECD 471, read across): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 100, TA 98, TA 102, E. coli WP2 uvrA, and E. coli WP2 uvrA pKM 101.
In vitro chromosome aberration in mammalian cells (OECD 473, read across): negative in Chinese hamster lung fibroblasts (V79) and in primary human peripheral lymphocytes with and without metabolic activation.
In vitro gene mutation in mammalian cells (OECD 476, read across): negative in Chinese hamster lung fibroblasts (V79) with and without metabolic activation.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 30 Jul - 07 Aug 2007
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- The analytical purity of the test substance was not reported.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted Jul 1997
- Deviations:
- yes
- Remarks:
- analytical purity of the test substance was not reported
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- yes
- Remarks:
- analytical purity of the test substance was not reported
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Direccion General de Farmacia y Productos Sanitarios, Consejera de Sanidad y Consumo, Madrid, Spain
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Metabolic activation system:
- Cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- 0.05, 0.15, 0.5, 1.5, 5 µL/plate, with and without metabolic activation. Purity of test substance was considered 100%.
Density of liquid test material was 0.854, no cytotoxicity was seen. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: no data
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- solvent type not reported
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-nitrofluorene, sodium azide, 9-aminoacridine, 4-nitroquinoline-N-oxide, 2-aminoanthracene
- Remarks:
- See 'Any other information on material and methods incl. tables' for details on positive controls
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); if the first assay result was negative the second assay was performed as a preincubation assay, and if the first assay result was positive the second assay was performed as a plate incorporation assay
DURATION
- Preincubation period: 20 min (second assay)
- Exposure duration: 48 hrs
NUMBER OF REPLICATIONS: 3 replications in 2 independent assays
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- A test substance is considered positive (mutagenic) in the test if:
It induces a dose-resonse in the range tested and/or
a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with ot without mutagenic activation is observed. - Statistics:
- Mean values and standard deviation were calculated.
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and 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:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- 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:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed at any dose level, with and without metabolic activation.
COMPARISON WITH HISTORICAL CONTROL DATA: yes, the negative control and test substance results fell within the historical control data range (see Table 1 under 'Any other information on material and methods incl. tables').
ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity was observed up to and including the highest dose level, which is the limit dose level recommended in the OECD 471 guideline.
OTHER: Only 2-amino anthracene was used as a positive control, however, dilutions of the sample S9-mix were tested and confirmed to activate benzo(a)pyrene and 2-amino anthracene. - Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 11 - 20 Mar 1998
- 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
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- 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:
- Cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- 10, 33, 100, 333 and 1000 µg/plate, with and without metabolic activation
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide, 9-aminoacridine, daunomycine, methylmethanesulfonate, 4-nitroquinoline-N-oxide, 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3 replications in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (reduction of bacterial background lawn, increase in size of the microcolonies, reduction of the revertant colonies)
OTHER:
the results of the dose range-finding test were included as part of experiment 1 - Evaluation criteria:
- A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following
criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain (see Table 1)
b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants compared to the solvent control value in any of the tester strains, either with or without metabolic activation. However, any plate with a mean plate count of less than 20 colonies is considered to be not significant and the result will be disregarded.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision. - Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed in all strains, with and without metabolic activation, from 1000 µg/plate and above.
RANGE-FINDING/SCREENING STUDIES:
A dose range-finding test with strain TA100 and the WP2uvrA strain (both with and without S9- mix) was performed to select suitable doses for the main experiments. Eight concentrations (3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate) were tested in triplicate. The results were reported as a part of the first experiment of the mutation assay. The highest concentration of the test substance used in the main experiments was the level at which the test substance exhibited limited solubility. Precipitation was observed on the plates from 1000 µg/plate and above in both strains.
COMPARISON WITH HISTORICAL CONTROL DATA: yes, the spontaneous mutation rate of each tester strain per plate were within the characteristic spontaneous mutation range (see Table 1 under 'Any other information on materials and methods incl. tables'), with the exception of TA 100 without metabolic activation. The value was slightly outside the limit of the range, therefore the validity of the test was considered not to be affected.
ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity was observed at any concentration level, with or without metabolic activation (see Table 2 and 3). - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 08 Mar - 07 Apr 1994
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted May 1983
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hessisches Ministerium für Umwelt, Energie und Bundesangelegenheiten, Wiesbaden, Germany
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: minimal essential medium (MEM) supplemented with 10 % fetal calf serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no
- doubling time of clone V79/T5 in stock cultures: 12 h - Metabolic activation:
- with and without
- Metabolic activation system:
- Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 500 mg/kg bw Aroclor 1254 i.p. 5 days prior to sacrifice.
- Test concentrations with justification for top dose:
- Experiment 1, chromosome aberration and mitotic index:
4 h treatment, 18 h harvest time, without metabolic activation: 10, 60 and 100 μg/mL
4 h treatment, 28 h harvest time, without metabolic activation: 100 μg/mL
18 h treatment, 18 h harvest time, with metabolic activation: 10, 60 and 100 μg/mL
28 h treatment, 28 h harvest time, with metabolic activation: 100 μg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol. The final concentration of ethanol in the culture medium did not exceed 1 % v/v.
- Justification for choice of solvent/vehicle: the solvent was chosen due to its solubility properties and its non-toxicity to the cells. - Untreated negative controls:
- yes
- Remarks:
- culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: ethylmethanesulphonate (600 µg/mL in nutrient medium, -S9) and cyclophosphamide (0.47 µg/mL in nutrient medium, +S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4h with metabolic activation, 18 and 28 h without metabolic activation
- Expression time (cells in growth medium): Preparation interval was 18 and 28 hours for all experiments. Therefore, for the cells exposed for 4 h with metabolic activation, the medium was changed to complete medium after the exposure for the remaining time up to preparation. The cells treated without metabolic activation were kept for the whole exposure duration of 18 or 28 hours in medium containing the test substance.
- Fixation time (start of exposure up to fixation or harvest of cells): 2.5 h after adding colcemid (at 18 and 28 h)
SPINDLE INHIBITOR (cytogenetic assays): 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/mL)
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: duplicates
NUMBER OF CELLS EVALUATED: at least 100 well spread metaphases per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: yes, the number of polyploid cells was scored (% polyploid metaphases) - Evaluation criteria:
- The test substance is considered as mutagenic, if it includes either a concentration-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points.
The test substance neither producing a concentration-related increase in the number of structural chromosomal aberrations nor a significant and positive response at any of the test points is considered non-mutagenic in this system. - Statistics:
- Chi-square test at five percent level (p < 0.05)
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Due to limited solubility of the test substance higher concentrations than 100 µg/mL for the cytogenetic evaluation were not feasible.
- Precipitation: observed at concentrations higher than 100 µg/mL
RANGE-FINDING/SCREENING STUDIES/ADDITIONAL INFORMATION ON CYTOTOXICITY:
A pre-test was performed to determine the toxicity of the test article, however the result could only be assessed qualitatively. The highest attainable concentration was 100 µg/mL, at which slight precipitation occured. No cytotoxicity was observed.
TEST CONCENTRATIONS
6 test concentrations (1, 3, 10, 30, 60 and 100 µg/mL) were listed in the study report under 'materials and method', while the results of 3 concentrations (10, 60 and 100 µg/mL) were reported.
COMPARISON WITH HISTORICAL CONTROL DATA: yes (data not presented)
In the absence and presence of S9-mix, the mitotic indices were not substantially reduced after treatment with the highest evaluated concentration at each fixation interval. In the cytogenetic experiment, at both fixation intervals in the absence and presence of S9-mix, the test material did not increase the frequency of cells with aberrations. No biologically relevant increase of polyploid metaphases as compared to the rates of the controls was found after treatment with the test material.The positive controls were valid (see Table 1 and 2) . - Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 11 Feb - 20 Apr 1998
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: cultured human peripheral lymphocytes
- Details on mammalian cell type (if applicable):
- - Type and identity of media: F10 complete culture medium, containing Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively), sodium bicarbonate (1.2 g/L) and 30 U/mL heparin.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Experiment 1:
24 h treatment, 24 h harvest time, without metabolic activation: 100, 333 and 1000 µg/mL
48 h treatment, 48 h harvest time, without metabolic activation: 1000 µg/mL
3 h treatment, 24 h harvest time, with metabolic activation: 100, 333 and 1000 µg/mL
3 h treatment, 48 h harvest time, with metabolic activation: 1000 µg/mL
Experiment 2:
24 h treatment, 24 h harvest time, without metabolic activation: 100, 333 and 1000 µg/mL
3 h treatment, 24 h harvest time, with metabolic activation: 100, 333 and 1000 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.9% DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: mitomycine C (0.2 µg/mL, 24 h treatment time, -S9; 0.1 µg/mL, 48 h treatment time, -S9) and cyclophosphamide (15 µg/mL in nutrient medium, 3 h treatment time, +S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 h with metabolic activation, 24 and 48 h without metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 h fixation time with and without metabolic activation
SPINDLE INHIBITOR (cytogenetic assays): 0.5 µg/mL colcemid, added 3 hours before harvest time (21 h with metabolic activation, 21 and 45 h without metabolic activation)
STAIN (for cytogenetic assays): 5% (v/v) Gimsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 200 (100 per culture, duplicate cultures)
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
OTHER:
blood samples were taken from healthy, adult male volunteers by venapuncture, using a sterile vessel containing sodium heparine. The blood samples were stored at 4-25 ºC. Cultures were started within 4 h after blood collection. The average generation time of the cells is 13.6-14.1 h. Cultures were incubated at 37 ºC for 48 h prior to exposure to the test substance. - Evaluation criteria:
- A chromosome aberration test was considered acceptable if it met the following criteria:
a) The numbers of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range (min = 0, max = 5 (mean = 0.9, standard deviation = 1.0) aberrant cells per 100 metaphases in the absence of S9-mix; gaps excluded and min = 0, max = 5 (mean = 0.7, standard deviation = 0.9) aberrant cells per 100 metaphases in the presence of S9-mix; gaps excluded)
b) The positive control substances should produce a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
c) A homogeneous response between the replicate cultures is observed.
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
b) A statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision. - Statistics:
- The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics.
- Key result
- Species / strain:
- lymphocytes: cultured human peripheral lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the culture medium at 1000 µg/mL.
RANGE-FINDING/SCREENING STUDIES:
A range-finding test was performed to select the dose levels for the main experiment, using 10, 33, 100, 333 and 1000 µg/mL, with and without metabolic activation. The test substance precipitated in the culture medium at 1000 µg/mL (see Table 1).
ADDITIONAL INFORMATION ON CYTOTOXICITY:
no cytotoxicity was observed at any concentration, with or without metabolic activation (see Table 2 and 3). - Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 08 Mar - 15 Apr 1994
- 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:
- adopted April 1984
- 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 OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hessisches Ministerium für Umwelt, Energie und Bundesangelegenheiten, Wiesbaden, Germany
- 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: minimal essential medium (MEM) supplemented with 10 % fetal calf serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes, by treatment with HAT-medium
- Doubling time 12 - 16 h in stock cultures - Metabolic activation:
- with and without
- Metabolic activation system:
- Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 500 mg/kg bw Aroclor 1254 i.p. 5 days prior to sacrifice.
- Test concentrations with justification for top dose:
- 10, 30, 60 and 100 µg/mL, with and without metabolic activation
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol. The final concentration of ethanol in the culture medium did not exceed 1 % v/v.
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its non-toxicity to the cells. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: ethylmethanesulphonate (600 µg/mL in MEM without FCS, -S9) and 7,12-dimethylbenz(a)anthracene (3.85 µg/mL in DMSO, +S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent):
Cloning efficiency: 7 days
Mutant selection: 9 and 12 days (experiments 2 and 1, respectively)
- Fixation time (start of exposure up to fixation or harvest of cells):
Cloning efficiency: 7 and 14 days
Mutant selection: 16 and 19 days (experiments 2 and 1, respectively)
Counting of colonies: the colonies were stained with 10 % methylene blue in 0.01 % KOH solution. Stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
SELECTION AGENT (mutation assays): thioguanine, 11 µg/mL medium (Sigma, Deisenhofen, Germany)
NUMBER OF REPLICATIONS: 1 replication in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: determination of concentration-related cloning efficiency on day 7 after start of exposure; determination of cell survival on day 14 after start of exposure (end of selection time) - Evaluation criteria:
- The test substance is considered to be positive if it induces either a concentration-related increase of the mutant frequency or a reproducible positive response for one of the test points.
A test material producing neither a concentration-related increase in the mutant frequency nor a reproducible positive response in any of the test points is considered non-mutagenic in this system.
A significant response is:
1) test substance induces reproducibly with one of the concentrations a mutation frequency that is 3 times higher than the spontaneous mutation frequency in the experiment
2) test substance induces reproducible concentration-related increase of the mutation frequency. May also be considered in case a 3-fold increase of the mutant frequency is not observed.
In a case-by-case evaluation the decision depends on the level of the corresponding negative control data. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- precipitation at limit of water solubility at 100 µg/mL observed.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Due to limited solubility of the test substance higher concentrations than 100 µg/mL for the cytogenetic evaluation were not feasible.
- Precipitation: observed at concentrations higher than 100 µg/mL
RANGE-FINDING/SCREENING STUDIES
Data on the pre-test were obtained from the chromosomal aberration study (IUCLID section 7.6.1: key, Emery, 1994, ChrAb, RL1)
A pre-test was performed to determine the toxicity of the test article. The test substance was dissolved in ethanol due to its solubility properties. The highest attainable concentration was 100 µg/mL, at which a slight precipitation was observed. No toxic effects were observed up to and including the highest dose level.
COMPARISON WITH HISTORICAL CONTROL DATA: yes (data not presented)
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In parallel to the mutagenicity testing the cloning efficiency was tested. Without metabolic activation the relative cloning efficiencies were between 64.2 - 91.8 % in treated cells compared to control cells. With metabolic activation the relative cloning efficiencies were between 92.4 - 109.7 % in treated cells compared to control cells.
No statistically significant increase in gene mutations was observed (see Table 1 and 2). The positive controls were shown to be valid. - Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Referenceopen allclose all
Table 1. Test results of experiment 1 (plate incorporation method)
With or without S9-Mix |
Test substance concentration (μL/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2 uvR |
TA98 |
TA1537 |
||
- |
Solvent |
49.7 ± 26.1 |
14.7 ± 1.5 |
76.0 ± 8.2 |
14.7 ± 2.5 |
27.3 ± 5.5 |
- |
0.05 |
41.0 ± 4.0 |
14.7 ± 3.8 |
77.3 ± 22.9 |
21.3 ± 3.8 |
28.0 ± 8.9 |
- |
0.15 |
37.7 ± 3.8 |
11.0 ± 1.0 |
59.7 ± 7.2 |
16.0 ± 7.1 |
29.0 ± 3.0 |
- |
0.5 |
36.3 ± 6.8 |
13.3 ± 3.1 |
59.7 ± 4.9 |
20.0 ± 4.6 |
40.0 ± 21.0 |
- |
1.5 |
42.7 ± 4.7 |
12.0 ± 2.6 |
55.7 ± 21.1 |
16.3 ± 4.9 |
34.3 ± 9.6 |
- |
5.0 |
47.3 ± 10.1 |
10.3 ± 2.1 |
43.3 ± 18.1 |
17.0 ± 1.0 |
37.3 ± 8.6 |
Positive controls, –S9 |
Name |
NaN3 |
NaN3 |
4 -NNO |
2-NF |
9-AA |
Concentrations positive controls (μg/plate) |
10 |
30 |
7 |
90 |
100 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
556.3 ± 55.2 |
184.7 ± 46.8 |
610.0 ± 30.3 |
399.3 ± 25.0 |
215.0 ± 1.0 |
|
+ |
Solvent |
44.0 ± 2.0 |
15.7 ± 7.4 |
71.7 ± 14.7 |
20.3 ± 4.7 |
8.7 ± 3.1 |
+ |
0.05 |
52.0 ± 6.1 |
14.3 ± 7.8 |
90.7 ± 17.5 |
27.7 ± 1.5 |
10.0 ± 5.6 |
+ |
0.15 |
67.0 ± 14.7 |
15.3 ± 5.1 |
76.0 ± 39.9 |
27.0 ± 5.3 |
5.7 ± 2.1 |
+ |
0.5 |
47.0 ± 10.1 |
15.7 ± 3.5 |
87.3 ± 29.9 |
26.3 ± 5.0 |
8.7 ± 0.6 |
+ |
1.5 |
49.7 ± 13.3 |
10.7 ± 1.2 |
80.7 ± 24.0 |
21.7 ± 4.5 |
9.0 ± 2.6 |
+ |
5.0 |
61.3 ± 9.0 |
10.7 ± 2.5 |
64.7 ± 29.7 |
19.7 ± 4.7 |
7.0 ± 4.6 |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μL/plate) |
10 |
30 |
7 |
10 |
30 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
305.0 ± 41.6 |
326.0 ± 10.8 |
320.0 ± 6.2 |
611.3 ± 108.1 |
320.0 ± 6.2 |
|
2-NF: 2-nitrofluorene
NaN3: sodium azide
9-AA: 9-aminoacridine
4-NNO: 4-nitroquinoline-N-oxide
2-AA: 2-aminoanthracene
Table 2. Test results of experiment 2 (preincubation method)
With or without S9-Mix |
Test substance concentration (μL/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard Deviation ) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2 uvR |
TA98 |
TA1537 |
||
- |
Solvent |
64.3 ± 5.1 |
9.7 ± 5.5 |
74.7 ± 19.0 |
22.7 ± 6.5 |
9.7 ± 3.2 |
- |
0.05 |
39.0 ± 1.0 |
12.3 ± 5.8 |
113.0 ± 15.7 |
23.3 ± 3.1 |
11.3 ± 3.1 |
- |
0.15 |
44.3 ± 4.2 |
12.3 ± 5.0 |
103.7 ± 17.6 |
24.0 ± 2.0 |
11.0 ± 3.5 |
- |
0.5 |
38.7 ± 14.2 |
9.0 ± 6.9 |
116.3 ± 10.8 |
22.7 ± 2.1 |
10.7 ± 1.5 |
- |
1.5 |
34.0 ± 3.0 |
16.7 ± 4.9 |
107.3 ± 10.1 |
29.3 ± 7.6 |
11.7 ± 5.5 |
- |
5.0 |
43.3 ± 10.7 |
10.0 ± 2.6 |
116.7 ± 10.1 |
19.3 ± 9.9 |
11.0 ± 5.2 |
Positive controls, –S9 |
Name |
NaN3 |
NaN3 |
4-NNO |
2-NF |
9-AA |
Concentrations (μL/plate) |
10 |
30 |
7 |
90 |
100 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
580.0 ± 8.2 |
504.0 ± 27.6 |
810.0 ± 77.3 |
544.3 ± 417.5 |
682.7 ± 91.5 |
|
+ |
Solvent |
47.0 ± 9.8 |
17.7 ± 5.5 |
45.3 ± 5.9 |
15.0 ± 4.0 |
19.3 ± 9.1 |
+ |
0.05 |
52.0 ± 6.6 |
18.0 ± 3.0 |
74.0 ± 5.6 |
17.3 ± 3.8 |
25.7 ± 1.5 |
+ |
0.15 |
51.3 ± 9.0 |
13.0 ± 7.9 |
80.0 ± 6.1 |
18.0 ± 4.4 |
24.7 ± 8.7 |
+ |
0.5 |
58.7 ± 8.5 |
16.0 ± 5.0 |
63.7 ± 14.5 |
17.7 ± 1.5 |
18.7 ± 3.1 |
+ |
1.5 |
58.7 ± 4.2 |
19.0 ± 6.2 |
76.0 ± 18.0 |
14.0 ± 3.6 |
18.3 ± 6.0 |
+ |
5.0 |
56.3 ± 4.1 |
15.7 ± 3.1 |
78.7 ± 26.1 |
16.0 ± 4.4 |
32.0 ± 6.9 |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μL/plate) |
10 |
30 |
7 |
10 |
30 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
352.7 ± 62.0 |
284.7 ± 11.7 |
475.7 ± 84.4 |
299.7 ± 79.6 |
161.7 ± 27.5 |
|
2-NF: 2-nitrofluorene
NaN3: sodium azide
9-AA: 9-aminoacridine
4-NNO: 4-nitroquinoline-N-oxide
2 -AA: 2 -aminoanthracene
Table 2: Results of experiment 1
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± SD) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA 1535 |
WP2 uvrA |
TA 98 |
TA 1537 |
||
-S9 |
ethanol |
55 ± 3 |
12 ± 3 |
14 ± 6 |
16 ± 2 |
6 ± 1 |
-S9 |
3 |
55 ± 6 |
- |
11 ± 2 |
- |
- |
-S9 |
10 |
63 ± 7 |
10 ± 4 |
10 ± 3 |
15 ± 6 |
7 ± 2 |
-S9 |
33 |
49 ± 7 |
8 ± 3 |
11 ± 1 |
16 ± 1 |
7 ± 4 |
-S9 |
100 |
59 ± 2 |
8 ± 1 |
12 ± 2 |
15 ± 3 |
5 ± 3 |
-S9 |
333 |
57 ± 7 |
7 ± 2 |
11 ± 6 |
17 ± 4 |
5 ± 3 |
-S9 |
1000 SP |
70 ± 6 |
10 ± 3 |
10 ± 2 |
16 ± 1 |
4 ± 1 |
-S9 |
3300 SP |
77 ± 9 |
- |
12 ± 2 |
- |
- |
-S9 |
5000 SP |
59 ± 13 |
- |
10 ± 4 |
- |
- |
Positive controls, –S9 |
Name |
MMS |
SA |
4-NQO |
DM |
9-AC |
Concentrations (μg/plate) |
650 |
1 |
10 |
4 |
60 |
|
|
529 ± 23 |
121 ± 18 |
291 ± 26 |
375 ± 36 |
185 ± 66 |
|
|
|
|
|
|
|
|
+S9 |
ethanol |
71 ± 3 |
8 ± 2 |
10 ± 1 |
26 ± 2 |
8 ± 1 |
+S9 |
3 |
66 ± 10 |
|
11 ± 4 |
|
|
+S9 |
10 |
62 ± 3 |
9 ± 2 |
9 ± 4 |
25 ± 4 |
8 ± 3 |
+S9 |
33 |
64 ± 3 |
9 ± 3 |
14 ± 3 |
22 ± 2 |
6 ± 2 |
+S9 |
100 |
50 ± 8 |
9 ± 3 |
15 ± 3 |
24 ± 5 |
6 ± 2 |
+S9 |
333 |
45 ± 5 |
13 ± 4 |
10 ± 3 |
18 ± 4 |
6 ± 4 |
+S9 |
1000 SP |
54 ± 8 |
12 ± 4 |
12 ± 6 |
23 ± 5 |
8 ± 1 |
+S9 |
3330 SP |
54 ± 9 |
- |
12 ± 1 |
- |
- |
+S9 |
5000 SP |
51 ± 9 |
- |
12 ± 2 |
- |
- |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
1 |
2.5 |
5 |
2.5 |
2.5 |
|
|
1182 ± 90 |
240 ± 8 |
88 ± 10 |
1047 ± 57 |
713 ± 149 |
|
MMS = methylmethanesulfonate
SA = sodium azide
4-NQO = 4-nitroquinoline-N-oxide
9-AC = 9-aminoacridine
DM = daunomycine
2-AA = 2-aminoanthracene
SP = slight precipitate
Table 3: Results of experiment 2
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± SD) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA 1535 |
WP2 uvrA |
TA 98 |
TA 1537 |
||
-S9 |
ethanol |
88 ± 10 |
10 ± 2 |
10 ± 6 |
22 ± 7 |
5 ± 2 |
-S9 |
10 |
94 ± 13 |
9 ± 3 |
9 ± 2 |
17 ± 2 |
7 ± 3 |
-S9 |
33 |
93 ± 3 |
8 ± 3 |
12 ± 2 |
17 ± 3 |
5 ± 1 |
-S9 |
100 |
90 ± 7 |
10 ± 3 |
9 ± 2 |
14 ± 5 |
5 ± 3 |
-S9 |
333 |
90 ± 9 |
6 ± 3 |
11 ± 3 |
15 ± 6 |
5 ± 1 |
-S9 |
1000 SP |
77 ± 5 |
6 ± 3 |
9 ± 4 |
14 ± 4 |
7 ± 4 |
Positive controls, –S9 |
Name |
MMS |
SA |
4-NQO |
DM |
9-AC |
Concentrations (μg/plate) |
650 |
1 |
10 |
4 |
60 |
|
|
714 ± 49 |
152 ± 10 |
936 ± 118 |
598 ± 16 |
251 ± 100 |
|
|
|
|
|
|
|
|
+S9 |
ethanol |
99 ± 8 |
8 ± 4 |
11 ± 7 |
15 ± 1 |
7 ± 3 |
+S9 |
10 |
98 ± 10 |
8 ± 3 |
7 ± 1 |
17 ± 3 |
4 ± 2 |
+S9 |
33 |
103 ± 21 |
6 ± 3 |
8 ± 2 |
27 ± 6 |
7 ± 1 |
+S9 |
100 |
98 ± 9 |
8 ± 3 |
13 ± 7 |
21 ± 7 |
6 ± 2 |
+S9 |
333 |
93 ± 6 |
7 ± 5 |
8 ± 3 |
20 ± 5 |
8 ± 2 |
+S9 |
1000 SP |
112 ± 2 |
9 ± 3 |
8 ± 1 |
23 ± 1 |
4 ± 1 |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
1 |
2.5 |
5 |
2.5 |
2.5 |
|
|
2235 ± 60 |
322 ± 15 |
406 ± 20 |
1732 ± 115 |
703 ± 41 |
|
MMS = methylmethanesulfonate
SA = sodium azide
4-NQO = 4-nitroquinoline-N-oxide
9-AC = 9-aminoacridine
DM = daunomycine
2-AA = 2-aminoanthracene
SP = slight precipitate
Table 1: Number of Polyploid Cells and Mitotic Index
|
conc. per mL |
Exposure duration (hours) |
S9 mix |
fixation interval |
mean number of polyploid cells* |
mitotic index** (%) |
neg. control |
|
4 |
- |
18 |
1.5 |
100.0 |
vehicle control |
1.0 % |
4 |
- |
18 |
1.5 |
100.0 |
EMS |
600 μg |
4 |
- |
18 |
3.0 |
68.9 |
test substance |
10 μg |
4 |
- |
18 |
1.0 |
83.4 |
test substance |
60 μg |
4 |
- |
18 |
2.5 |
100.0 |
test substance |
100 μg |
4 |
- |
18 |
2.0 |
89.7 |
neg. control |
|
18 |
+ |
18 |
2.0 |
100.0 |
vehicle control |
1.0 % |
18 |
+ |
18 |
3.0 |
100.0 |
CPA |
0.47 μg |
18 |
+ |
18 |
3.0 |
63.0 |
test substance |
10 μg |
18 |
+ |
18 |
1.5 |
127.1 |
test substance |
60 μg |
18 |
+ |
18 |
2.0 |
113.7 |
test substance |
100 μg |
18 |
+ |
18 |
2.0 |
104.3 |
vehicle control |
1.0 % |
4 |
- |
28 |
4.0 |
100.0 |
test substance |
100 μg |
4 |
- |
28 |
5.0 |
119.0 |
vehicle control |
1.0 % |
28 |
+ |
28 |
2.5 |
100.0 |
test substance |
100 μg |
28 |
+ |
28 |
2.5 |
91.5 |
* The number of polyploid cells was determined in 100 cells per culture of each test group.
** The mitotic index was determined in 1000 cells per culture of each test group.
Table 2: Structural chromosomal aberrations
|
conc. per mL |
S9 mix |
fixation interval |
cells scored |
aberrant cells (% mean) |
exchanges |
|
incl. gaps |
excl. gaps |
||||||
neg. control |
|
- |
18 |
200 |
0.0 |
0.0 |
0.0 |
vehicle control |
1.0 % |
- |
18 |
200 |
1.5 |
0.5 |
0.0 |
EMS |
600 μg |
- |
18 |
200 |
23.5 |
22.0 |
16.5 |
test substance |
10 μg |
- |
18 |
200 |
1.5 |
0.5 |
0.0 |
test substance |
60 μg |
- |
18 |
200 |
1.0 |
1.0 |
1.0 |
test substance |
100 μg |
- |
18 |
200 |
5.0 |
3.0 |
0.0 |
neg. control |
|
+ |
18 |
200 |
1.0 |
1.0 |
0.0 |
vehicle control |
1.0 % |
+ |
18 |
200 |
4.0 |
3.5 |
0.5 |
CPA |
0.47 μg |
+ |
18 |
200 |
23.0 |
22.0 |
11.0 |
test substance |
10 μg |
+ |
18 |
200 |
3.0 |
1.0 |
0.0 |
test substance |
60 μg |
+ |
18 |
200 |
2.0 |
2.0 |
1.5 |
test substance |
100 μg |
+ |
18 |
200 |
3.0 |
1.5 |
0.0 |
vehicle control |
1.0 % |
- |
28 |
200 |
4.0 |
2.0 |
0.0 |
test substance |
100 μg |
- |
28 |
200 |
3.5 |
2.0 |
0.0 |
vehicle control |
1.0 % |
+ |
28 |
200 |
4.0 |
2.5 |
0.5 |
test substance |
100 μg |
+ |
28 |
200 |
4.0 |
2.0 |
1.0 |
EMS = Ethylmethanesulphonate
CPA = Cyclophosphamide
Table 1: results of range-finding assay
Metabolic activation |
Test substance concentration (μg/mL) |
Mitotic index (%) |
|
|
|
24 h harvesting time |
48 h harvesting time |
- |
Vehicle control (DMSO) |
100 |
100 |
- |
10 |
91 |
98 |
- |
33 |
86 |
98 |
- |
100 |
91 |
105 |
- |
333 |
93 |
105 |
- |
1000 |
100 |
91 |
+ |
Vehicle control (DMSO) |
100 |
|
+ |
10 |
92 |
- |
+ |
33 |
90 |
- |
+ |
100 |
94 |
- |
+ |
333 |
98 |
- |
+ |
1000 |
94 |
- |
Table 1: Chromosome aberrations, summarised data, experiment 1
Metabolic activation |
Test substance concentration (μg/mL) |
Total number of metaphases analysed |
Total number of aberrations1 |
Number of cells with aberrations1 |
Mitotic index (%)2
|
||
|
|
|
Incl. gaps |
Excl. gaps |
Incl. gaps |
Excl. gaps |
|
24 h treatment, 24 h harvesting time |
|||||||
- |
Vehicle control (DMSO 1%) |
200 |
1 |
1 |
1 |
1 |
100 |
- |
100 |
200 |
7 |
6 |
6 |
5 |
98 |
- |
333 |
200 |
1 |
1 |
1 |
1 |
106 |
- |
1000 |
200 |
3 |
2 |
3 |
2 |
106 |
- |
mitomycin C |
200 |
41 |
37 |
38*** |
34*** |
61 |
48 h treatment, 48 h harvesting time |
|||||||
- |
Vehicle control (DMSO 1%) |
200 |
1 |
1 |
1 |
1 |
100 |
- |
1000 |
200 |
1 |
1 |
1 |
1 |
81 |
- |
mitomycin C |
150 |
72 |
65 |
59*** |
55*** |
49 |
3 h treatment, 24 h harvesting time |
|||||||
+ |
Vehicle control (DMSO 1%) |
200 |
4 |
2 |
4 |
2 |
100 |
+ |
100 |
200 |
4 |
4 |
4 |
4 |
68 |
+ |
333 |
200 |
0 |
0 |
0 |
0 |
81 |
+ |
1000 |
200 |
1 |
1 |
1 |
1 |
73 |
+ |
cyclophosphamide |
200 |
89 |
75 |
71*** |
63*** |
33 |
3 h treatment, 48 h harvesting time |
|||||||
+ |
Vehicle control (DMSO 1%) |
200 |
9 |
9 |
5 |
5 |
100 |
+ |
1000 |
200 |
1 |
1 |
1 |
1 |
81 |
1gaps include chromatid and isochromatid (chromosome) gaps
2percentage of metaphases per 1000 cells, compared to control
* P < 0.05; ** P < 0.01; *** P < 0.001 (Chi-square test)
Table 2: Chromosome aberrations, summarised data, experiment 2
Metabolic activation |
Test substance concentration (μg/mL) |
Total number of metaphases analysed |
Total number of aberrations1 |
Number of cells with aberrations1 |
Mitotic index (%)2
|
||
|
|
|
Incl. gaps |
Excl. gaps |
Incl. gaps |
Excl. gaps |
|
24 h treatment, 24 h harvesting time |
|||||||
- |
Vehicle control (DMSO 1%) |
200 |
0 |
0 |
0 |
0 |
100 |
- |
100 |
200 |
2 |
2 |
2 |
2 |
99 |
- |
333 |
200 |
4 |
4 |
4 |
4 |
113 |
- |
1000 |
200 |
1 |
1 |
1 |
1 |
97 |
- |
mitomycin C |
150 |
63 |
63 |
53*** |
53*** |
39 |
3 h treatment, 24 h harvesting time |
|||||||
+ |
Vehicle control (DMSO 1%) |
200 |
3 |
3 |
3 |
3 |
100 |
+ |
100 |
200 |
2 |
2 |
2 |
2 |
108 |
+ |
333 |
200 |
2 |
2 |
2 |
2 |
101 |
+ |
1000 |
200 |
2 |
2 |
2 |
2 |
104 |
+ |
Cyclophosphamide |
200 |
111 |
109 |
80*** |
79*** |
32 |
1gaps include chromatid and isochromatid (chromosome) gaps
2percentage of metaphases per 1000 cells, compared to control
* P < 0.05; ** P < 0.01; *** P < 0.001 (Chi-square test)
Table 1: mutagenicity data, experiment 1
|
Concentration (µg/mL) |
Metabolic activation |
No. of mutant colonies after plating in TG medium* (mean of 5 flasks) |
% cell survival |
No. of mutant colonies per 106 surviving cells |
% cloning efficiency |
Negative control |
- |
- |
2.2 |
54 |
9.4 |
100.0 |
Vehicle control |
- |
- |
1.0 |
66 |
3.9 |
100.0 |
EMS |
600 |
- |
89.6 |
50 |
416.4 |
73.8 |
Test substance |
10 |
- |
1.0 |
72 |
3.3 |
91.8 |
Test substance |
30 |
- |
2.0 |
72 |
6.8 |
72.0 |
Test substance |
60 |
- |
0.2 |
81 |
0.6 |
68.1 |
Test substance |
100 |
- |
2.8 |
65 |
10.2 |
64.2 |
Negative control |
- |
+ |
2.0 |
69 |
7.3 |
100.0 |
Vehicle control |
- |
+ |
3.2 |
68 |
11.4 |
100.0 |
DMBA |
3.85 |
+ |
112.0 |
57 |
496.2 |
53.1 |
Test substance |
10 |
+ |
5.4 |
78 |
16.1 |
92.4 |
Test substance |
30 |
+ |
0.2 |
65 |
0.8 |
95.6 |
Test substance |
60 |
+ |
1.4 |
67 |
5.4 |
97.3 |
Test substance |
100 |
+ |
0.2 |
74 |
0.7 |
109.7 |
*only colonies with more than 50 cells 8 days after seeding in TG selection medium were scored
** ration of mean number of cells found after 7 days in normal growth medium / cell number seeded per flask
TG = Thioguanine
EMS = Ethylmethanesulphonate
DMBA = 7,12-dimethylbenz(a)anthracene
Table 2: mutagenicity data, experiment 2
|
Concentration (µg/mL) |
Metabolic activation |
No. of mutant colonies after plating in TG medium* (mean of 5 flasks) |
% cell survival |
No. of mutant colonies per 106 surviving cells |
% cloning efficiency |
Negative control |
- |
- |
2.0 |
69 |
6.6 |
100.0 |
Vehicle control |
- |
- |
1.8 |
64 |
6.4 |
100.0 |
EMS |
600 |
- |
145.8 |
60 |
496.9 |
61.5 |
Test substance |
10 |
- |
4.2 |
62 |
18.4 |
93.6 |
Test substance |
30 |
- |
1.0 |
63 |
4.1 |
107.8 |
Test substance |
60 |
- |
3.2 |
60 |
13.5 |
56.4 |
Test substance |
100 |
- |
0.8 |
59 |
3.5 |
61.4 |
Negative control |
- |
+ |
2.4 |
60 |
10.3 |
100.0 |
Vehicle control |
- |
+ |
2.2 |
61 |
9.0 |
100.0 |
DMBA |
3.85 |
+ |
141.6 |
64 |
550.4 |
50.3 |
Test substance |
10 |
+ |
1.4 |
61 |
5.7 |
104.8 |
Test substance |
30 |
+ |
2.2 |
72 |
7.5 |
106.1 |
Test substance |
60 |
+ |
0.6 |
69 |
2.1 |
102.8 |
Test substance |
100 |
+ |
1.4 |
79 |
4.4 |
107.7 |
*only colonies with more than 50 cells 8 days after seeding in TG selection medium were scored
** ratio of mean number of cells found after 7 days in normal growth medium / cell number seeded per flask
TG = Thioguanine
EMS = Ethylmethanesulphonate
DMBA = 7,12-dimethylbenz(a)anthracene
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
No data on the potential for genetic toxicity of Hexadecyl palmitate (CAS 540-10-3) are available.
The assessment was therefore based on studies conducted with analogue substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).
Genetic toxicity (mutagenicity) in bacteria in vitro
CAS 93803-87-3
The in vitro genetic toxicity of 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) was assessed in a bacterial reverse mutation study (Ames test), performed under GLP conditions according to OECD guideline 471 (WoE, Ames, 1998). S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A were exposed to the test substance at concentrations up to 1000 µg/plate. Precipitation was observed in the medium at 1000 µg/plate and above in all strains, with and without metabolic activation. The negative and positive controls were valid. The test substance did not induce a significant increase in reversions in the S. typhimurium strains or E. coli strain, with or without metabolic activation.
CAS 22766-83-2
The in vitro genetic toxicity of 2-octyldodecyl myristate (CAS 22766-83-2) was assessed in a bacterial reverse mutation study (Ames test), performed under GLP conditions according to OECD guideline 471 (WoE, 2007). The plate incorporation method was applied in the first experiment and the preincubation method in the second experiment, using S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvrA pKM 101. All the strains were tested at concentrations up to the limit value of 5 µL/plate. . No cytotoxicity was observed, with and without metabolic activation. The positive and vehicle controls were valid. The test substance did not induce an increase in reversions in the S. typhimurium and E.coli strains, with or without metabolic activation.
Genetic toxicity (mutagenicity) in mammalian cells in vitro
CAS 3687-45-4
An in vitro mammalian cell gene mutation study was performed with Oleyl oleate (CAS 3687-45-4) under GLP conditions and according to OECD guideline 476 (WoE, 1994). Two separate experiments were performed. Chinese hamster lung fibroblast (V79) cells were treated with Oleyl oleate at concentrations of up to 100 µg/mL for 4 hours, with and without metabolic activation. After an expression time of 7 days in growth medium, cells were incubated for 9 or 12 days with 6-thioguanine as selection agent for forward mutation at the HPRT locus. Precipitation was seen at concentrations of 100 µg/mL and higher, while no cytotoxicity was observed at any concentration level. The positive and negative controls were valid and within the range of historical control data. No significant increase in mutation frequency was observed, with and without metabolic activation.
Genetic toxicity (cytogenicity) in mammalian cells in vitro
CAS 3687-45-4
The potential of Oleyl oleate (CAS 3687-45-4) to induce chromosomal aberrations was assessed using Chinese hamster lung fibroblast (V79) cells, in a GLP study performed according to OECD 473 (WoE, CA, 1994). The V79-cells were exposed to Oleyl oleate at concentrations up to 100 µg/mL, with and without metabolic activation (S9-mix). One experiment with duplicate replications was performed. A 4-hour treatment was performed without metabolic activation, using 10, 60 and 100 µg/mL concentration levels with 18-hour fixation time; and a 100 µg/mL concentration level with a 28-hour fixation time. The treatment with metabolic activation was performed at concentrations of 10, 60 and 100 µg/mL with an 18-hour treatment time, and 18-hour fixation time; and at 100 µg/mL with a 28-hour treatment time and 28-hour fixation time, respectively. Precipitation was observed at concentrations from 100 µg/mL, while no cytotoxicity was noted at any concentration. The negative and positive controls were valid.The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, with or without metabolic activation.
CAS 93803-87-3
The cytogenetic potential of 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) was assessed in an in vitro mammalian chromosome aberration test in primary human lymphocytes, performed according to OECD guideline 473 and under GLP conditions (WoE, CA, 1998). Duplicate cultures of cultured human peripheral lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation. In the first experiment, cells were incubated with test substance concentrations of 100, 333 and 1000 µg/mL for 24 hours with a 24-hour fixation time and at 1000 µg/mL for 48 hours with a 48-hour fixation time, in the absence of a metabolic activation system. The first experiment was also performed with cells exposed to 100, 333 and 1000 µg/mL for 3 hours with a 24-hour fixation time, and at 1000 µg/mL for 3 hours with a 48-hour fixation time in the presence of metabolic activation. In the second experiment cells were incubated with 100, 333 and 1000 µg/mL for 24 hours followed by a 24-hour expression time, without metabolic activation. In the presence of metabolic activation cell were exposed to 100, 333 and 1000 µg/mL for 3 hours followed by a 24-hour expression time. No cytotoxicity was observed. At 1000 µg/mL, precipitation was observed in the culture medium. The vehicle and positive controls were valid. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, with or without metabolic activation.
Overall conclusion for genetic toxicity
There are no available studies on the genetic toxicity of the target substance Hexadecyl palmitate (CAS 540-10-3). Therefore analogue read-across from source substances was applied from bacterial reverse mutation assays, in vitro studies on cytogenicity, and in vitro studies on gene mutation in mammalian cells. The results of the available in vitro studies were consistently negative. Based on the available data and following the analogue approach, no mutagenic or clastogenic potential in vitro is expected for Hexadecyl palmitate (CAS 540-10-3).
Justification for classification or non-classification
According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Hexadecyl palmitate (CAS 540-10-3), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.
Therefore, based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.
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