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EC number: 269-642-9 | CAS number: 68308-30-5
Toxicological information
Genetic toxicity: in vitro
Currently viewing:
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 09 Oct 2012 - 10 Feb 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
- Report date:
- 2013
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted Jul 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- 1998
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- National Institute for Quality and Organisational Development in Healthcare and Medicines, Budapest, Hungary
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Fatty acids, montan-wax, stearyl esters
- EC Number:
- 269-642-9
- EC Name:
- Fatty acids, montan-wax, stearyl esters
- Cas Number:
- 68308-30-5
- Molecular formula:
- C18 H38 O - C66 H130 O4
- IUPAC Name:
- Fatty acids C22-30 (even numbered), octadecyl alcohol esters
Constituent 1
Method
- Target gene:
- his operon; trp operon
Species / strainopen allclose all
- 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 phenobarbital and β-naphthoflavone
- Test concentrations with justification for top dose:
- Range finding test for toxicity:
10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation in TA100 and TA98
Based on the results of the range finding test the following concentrations were chosen for the main test:
First experiment: 5, 15.81, 50, 158.1, 500, 1581 snd 5000 µg/plate with and without metabolic activation
Second experiment: 5, 15.81, 50, 158.1, 500, 1581 snd 5000 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Solvent used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent: The test substance was insoluble in distilled water and acetone. Partial dissolution was observed in dimethylformamide, DMSO, n-Hexane and Ethanol at 100 mg/mL concentrations, however the formulations at 50 mg/mL using DMSO and DMF as vehicles after an approximately 3-minute incubation in an ultrasonic water bath were suitable for the test. Due to the better biocompatibility to the test system, DMSO was selected for vehicle of the study.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-1,2-phenylene-diamine: -S9: 4 µg/plate for TA98; 2-aminoanthracene: +S9: 2 or 50 µg/plate for all strains
- Remarks:
- The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-aminoanthracene and benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 min (preincubation method)
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: Reduction of the number of revertant colonies and background lawn - Evaluation criteria:
- The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test substance-treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor (= mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate) values were calculated for each concentration level of the test item and for the controls using Microsoft Excel TM software.
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- in all strains: the number of reversion was more than twice higher than the reversion rate of the negative (vehicle/solvent) control.
Statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- 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:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium 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:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at the highest tested concentration of 5000 µg/plate in all tester strains with and without metabolic activation.
RANGE-FINDING/SCREENING STUDIES: In the range finding test, only a slight inhibitory, cytotoxic effect of the test substance (slightly reduced background lawn development) was detected on one plate in Salmonella typhimurium TA100 tester strain at 5000 μg/plate concentration without metabolic activation. Based on the results of this test, the test substance was tested up to 5000 µg/plate with and wothout metabolic activation in all tester strains.
HISTORICAL CONTROL DATA
- Positive historical control data: The positive control values were within the range of the historical control data and therefore considered to be valid (please refer to table 1 and 2 under "any other information on materials and results incl. tables").
- Negative historical control data: The negative (untreated, DMSO) control values were within the range of the historical control data and therefore considered to be valid (please refer to table 3 under "any other information on materials and results incl. tables").
Any other information on results incl. tables
Table 4: Summary of the range finding test
| Concentrations (µg/plate) | Mean values of revertants / Mutation factor (MF) | Salmonella typhimurium tester strains | |||
| TA98 | TA100 | ||||
| -S9 | +S9 | -S9 | +S9 | ||
| Untreated control | Mean | 25.7 | 29.3 | 111.0 | 127.3 |
| MF | 1.33 | 1.31 | 0.76 | 0.96 | |
| Distilled water control | Mean | - | - | 124.0 | - |
| MF | - | - | 0.84 | - | |
| DMSO control (50 µL) | Mean | 21.0 | 36.3 | - | 210.7 |
| MF | 1.09 | 1.63 | - | 1.58 | |
| DMSO control (100 µL) | Mean | 19.3 | 22.3 | 147.0 | 133.0 |
| MF | 1.00 | 1.00 | 1.00 | 1.00 | |
| 5000 | Mean | 20.7 | 34.7 | 111.0 | 143.3 |
| MF | 1.07 | 1.55 | 0.76 | 1.08 | |
| 2500 | Mean | 22.7 | 26.0 | 140.3 | 162.3 |
| MF | 1.17 | 1.16 | 0.95 | 1.22 | |
| 1000 | Mean | 24.3 | 31.0 | 138.3 | 167.7 |
| MF | 1.26 | 1.39 | 0.94 | 1.26 | |
| 316 | Mean | 22.3 | 28.7 | 138.3 | 173.0 |
| MF | 1.16 | 1.28 | 0.94 | 1.30 | |
| 100 | Mean | 21.0 | 31.0 | 147.3 | 170.3 |
| MF | 1.09 | 1.39 | 1.00 | 1.28 | |
| 31.6 | Mean | 25.7 | 30.7 | 132.3 | 158.7 |
| MF | 1.33 | 1.37 | 0.90 | 1.19 | |
| Mean | 25.3 | 26.3 | 145.7 | 143.7 | |
| 10 | MF | 1.31 | 1.18 | 0.99 | 1.08 |
| NPD (4µg) | Mean | 289.3 | - | - | - |
| MF | 13.78 | - | - | - | |
| 2AA (2 µg) | Mean | - | 2400.0 | - | 2273.3 |
| MF | - | 66.06 | - | 10.79 | |
| SA (2 µg) | Mean | - | - | 1244.0 | - |
| MF | - | - | 10.03 | - | |
NPD: 4-nitro-1,2-phenylene- diamine
2AA: 2-aminoanthracene
SA: Sodium azide
MF: Mutation factor
Table 5: Summary of Experiment I (plate incorporation)
| Concentrations (µg/plate) | Mean values of revertants / Mutation factor (MF) | Salmonella typhimurium tester strains | Escherichia coli | ||||||||
| TA98 | TA100 | TA1535 | TA1537 | WP2 uvrA | |||||||
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | ||
| Untreated control | Mean | 24.3 | 33.0 | 99.0 | 120.3 | 4.3 | 7.7 | 7.7 | 7.7 | 28.0 | 37.0 |
| MF | 0.99 | 1.38 | 0.97 | 1.08 | 0.59 | 1.10 | 0.96 | 1.44 | 0.80 | 1.05 | |
| Distilled water control | Mean | - | - | 100.3 | - | 4.7 | - | - | - | 35.7 | - |
| MF | - | - | 0.99 | - | 0.64 | - | - | - | 1.02 | - | |
| DMSO control (50 µL) | Mean | 24.0 | 31.7 | - | 112.7 | - | 7.3 | 8.3 | 6.3 | - | 44.3 |
| MF | 0.97 | 1.32 | - | 1.01 | - | 1.05 | 1.04 | 1.19 | - | 1.25 | |
| DMSO control (100 µL) | Mean | 24.7 | 24.0 | 101.7 | 111.7 | 7.3 | 7.0 | 8.0 | 5.3 | 35.0 | 35.3 |
| MF | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | |
| 5000 | Mean | 27.7 | 29.7 | 115.0 | 134.0 | 7.3 | 13.7 | 6.7 | 10.3 | 36.3 | 34.0 |
| MF | 1.12 | 1.24 | 1.13 | 1.20 | 1.00 | 1.95 | 0.83 | 1.94 | 1.04 | 0.96 | |
| 1581 | Mean | 27.7 | 29.0 | 99.0 | 125.3 | 6.7 | 9.3 | 8.0 | 7.3 | 30.7 | 37.3 |
| MF | 1.12 | 1.21 | 0.97 | 1.12 | 0.91 | 1.33 | 1.00 | 1.38 | 0.88 | 1.06 | |
| 500 | Mean | 24.3 | 26.0 | 103.3 | 124.0 | 8.3 | 13.0 | 6.0 | 8.3 | 37.3 | 40.0 |
| MF | 0.99 | 1.08 | 1.02 | 1.11 | 1.14 | 1.86 | 0.75 | 1.56 | 1.07 | 1.13 | |
| 158.1 | Mean | 21.7 | 25.0 | 104.3 | 126.7 | 8.0 | 11.3 | 5.0 | 8.7 | 34.3 | 34.0 |
| MF | 0.88 | 1.04 | 1.03 | 1.13 | 1.09 | 1.62 | 0.63 | 1.63 | 0.98 | 0.96 | |
| 50 | Mean | 21.7 | 25.3 | 101.7 | 129.0 | 6.0 | 11.0 | 4.0 | 8.0 | 34.3 | 30.7 |
| MF | 0.88 | 1.06 | 1.00 | 1.16 | 0.82 | 1.57 | 0.50 | 1.50 | 0.98 | 0.87 | |
| 15.81 | Mean | 18.3 | 26.7 | 123.3 | 128.7 | 13.3 | 9.7 | 7.0 | 9.7 | 30.7 | 30.0 |
| MF | 0.74 | 1.11 | 1.21 | 1.15 | 1.82 | 1.38 | 0.88 | 1.81 | 0.88 | 0.85 | |
| 5 | Mean | 21.0 | 30.3 | 112.0 | 122.3 | 6.7 | 9.7 | 7.0 | 9.3 | 28.3 | 40.3 |
| MF | 0.85 | 1.26 | 1.10 | 1.10 | 0.91 | 1.38 | 0.88 | 1.75 | 0.81 | 1.14 | |
| NPD (4 µg) | Mean | 315.3 | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| MF | 13.14 | -- | -- | -- | -- | -- | -- | -- | -- | -- | |
| 2AA (2 µg) | Mean | -- | 2282.7 | -- | 2111.3 | -- | 208.3 | -- | 208.3 | -- | -- |
| MF | -- | 72.08 | -- | 18.74 | -- | 28.41 | -- | 32.89 | -- | -- | |
| 2AA (50 µg) | Mean | -- | -- | -- | -- | -- | -- | -- | -- | -- | 247.7 |
| MF | -- | -- | -- | -- | -- | -- | -- | -- | -- | 5.59 | |
| SA (2 µg) | Mean | -- | -- | 1210.7 | -- | 1247.7 | -- | -- | -- | -- | -- |
| MF | -- | -- | 12.07 | -- | 266.29 | -- | -- | -- | -- | -- | |
| 9AA (50 µg) | Mean | -- | -- | -- | -- | -- | -- | 498.3 | -- | -- | -- |
| MF | -- | -- | -- | -- | -- | -- | 59.80 | -- | -- | -- | |
| MMS (2 µL) | Mean | -- | -- | -- | -- | -- | -- | -- | -- | 1203.3 | -- |
| MF | -- | -- | -- | -- | -- | -- | -- | -- | 33.74 | -- | |
NPD: 4-nitro-1,2-phenylene- diamine
2AA: 2-aminoanthracene
SA: Sodium azide
9AA: 9-aminoacridine
MMS: Methylmethanesulfonate
MF: Mutation factor
Table 6: Summary of Experiment II (preincubation method)
| Concentrations (µg/plate) | Mean values of revertants / Mutation factor (MF) | Salmonella typhimurium tester strains | Escherichia coli | ||||||||
| TA98 | TA100 | TA1535 | TA1537 | WP2 uvrA | |||||||
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | ||
| Untreated control | Mean | 23.3 | 40.7 | 105.3 | 142.0 | 5.7 | 11.3 | 7.0 | 11.0 | 34.7 | 50.3 |
| MF | 1.43 | 1.49 | 0.98 | 1.20 | 0.71 | 0.83 | 1.11 | 1.43 | 1.05 | 1.00 | |
| Distilled water control | Mean | - | - | 115.7 | - | 15.7 | - | - | - | 42.3 | - |
| MF | - | - | 1.08 | - | 1.96 | - | - | - | 1.28 | - | |
| DMSO control (50 µL) | Mean | 20.7 | 34.0 | - | 114.7 | - | 13.7 | 9.3 | 10.3 | - | 48.7 |
| MF | 1.27 | 1.24 | - | 0.97 | - | 1.00 | 1.47 | 1.35 | - | 0.97 | |
| DMSO control (100 µL) | Mean | 16.3 | 27.3 | 107.3 | 118.0 | 8.0 | 13.7 | 6.3 | 7.7 | 33.0 | 50.3 |
| MF | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | |
| 5000 | Mean | 42.0 | 45.0 | 117.7 | 129.3 | 11.0 | 11.0 | 9.3 | 16.0 | 34.0 | 55.5 |
| MF | 2.57 | 1.65 | 1.10 | 1.10 | 1.38 | 0.80 | 1.47 | 2.09 | 1.03 | 1.10 | |
| 1581 | Mean | 27.0 | 42.7 | 113.0 | 119.7 | 6.7 | 8.7 | 7.7 | 10.3 | 38.7 | 53.7 |
| MF | 1.65 | 1.56 | 1.05 | 1.01 | 0.83 | 0.63 | 1.21 | 1.35 | 1.17 | 1.07 | |
| 500 | Mean | 27.0 | 33.3 | 112.7 | 120.3 | 7.7 | 8.7 | 8.7 | 15.0 | 38.0 | 50.3 |
| MF | 1.65 | 1.22 | 1.05 | 1.02 | 0.96 | 0.63 | 1.37 | 1.96 | 1.15 | 1.00 | |
| 158.1 | Mean | 24.0 | 32.0 | 118.3 | 125.7 | 7.3 | 9.3 | 8.7 | 10.0 | 33.0 | 56.7 |
| MF | 1.47 | 1.17 | 1.10 | 1.06 | 0.92 | 0.68 | 1.37 | 1.30 | 1.00 | 1.13 | |
| 50 | Mean | 26.7 | 27.0 | 104.0 | 121.3 | 9.0 | 10.7 | 6.0 | 6.7 | 41.7 | 56.7 |
| MF | 1.63 | 0.99 | 0.97 | 1.03 | 1.13 | 0.78 | 0.95 | 0.87 | 1.26 | 1.13 | |
| 15.81 | Mean | 22.0 | 30.3 | 107.0 | 127.7 | 6.7 | 9.3 | 6.3 | 9.3 | 32.7 | 54.3 |
| MF | 1.35 | 1.11 | 1.00 | 1.08 | 0.83 | 0.68 | 1.00 | 1.22 | 0.99 | 1.08 | |
| 5 | Mean | 30.7 | 26.3 | 121.7 | 125.0 | 7.3 | 8.7 | 3.7 | 11.0 | 38.3 | 48.0 |
| MF | 1.88 | 0.96 | 1.13 | 1.06 | 0.92 | 0.63 | 0.58 | 1.43 | 1.16 | 0.95 | |
| NPD (4 µg) | Mean | 392.7 | - | - | - | - | - | - | - | - | - |
| MF | 19.0 | - | - | - | - | - | - | - | - | - | |
| 2AA (2 µg) | Mean | - | 2404.0 | - | 2444.0 | - | 204.3 | - | 201.3 | - | - |
| MF | - | 70.71 | - | 21.31 | - | 14.95 | - | 19.48 | - | - | |
| 2AA (50 µg) | Mean | - | - | - | - | - | - | - | - | - | 285.0 |
| MF | - | - | - | - | - | - | - | - | - | 5.86 | |
| SA (2 µg) | Mean | - | - | 1385.3 | - | 1169.3 | - | - | - | - | - |
| MF | - | - | 11.98 | - | 74.64 | - | - | - | - | - | |
| 9AA (50 µg) | Mean | - | - | - | - | - | - | 380.7 | - | - | - |
| MF | - | - | - | - | - | - | 40.79 | - | - | - | |
| MMS (2 µL) | Mean | - | - | - | - | - | - | - | - | 1098.7 | - |
| MF | - | - | - | - | - | - | - | - | 25.95 | - | |
NPD: 4-nitro-1,2-phenylene- diamine
2AA: 2-aminoanthracene
SA: Sodium azide
9AA: 9-aminoacridine
MMS: Methylmethanesulfonate
MF: Mutation factor
Table 6: Summary of the repeated Experiment II (pre-experiment)
| Concentrations (µg/plate) | Mean values of revertants / Mutation factor (MF) | Salmonella typhimurium tester strains | |||
| TA98 | TA1537 | ||||
| -S9 | +S9 | -S9 | +S9 | ||
| Untreated control | Mean | 28.3 | - | - | 8.0 |
| MF | 1.27 | - | - | 1.26 | |
| Distilled water control | Mean | 25.3 | - | - | 8.7 |
| MF | 1.13 | - | - | 1.37 | |
| DMSO control (50 µL) | Mean | 22.3 | - | - | 6.3 |
| MF | 1.00 | - | - | 1.00 | |
| DMSO control (100 µL) | Mean | 25.7 | - | - | 5.3 |
| MF | 1.15 | - | - | 0.84 | |
| 5000 | Mean | 24.0 | - | - | 5.7 |
| MF | 1.07 | - | - | 0.89 | |
| 1581 | Mean | 23.3 | - | - | 5.3 |
| MF | 1.04 | - | - | 0.84 | |
| 500 | Mean | 23.3 | - | - | 7.0 |
| MF | 1.04 | - | - | 1.11 | |
| 158.1 | Mean | 20.7 | - | - | 8.3 |
| MF | 0.93 | - | - | 1.32 | |
| 50 | Mean | 24.0 | - | - | 7.0 |
| MF | 1.07 | - | - | 1.11 | |
| 15.81 | Mean | 21.3 | - | - | 5.0 |
| MF | 0.96 | - | - | 0.79 | |
| 5 | Mean | 18.3 | - | - | 6.7 |
| MF | 0.82 | - | - | 1.05 | |
| NPD (4 µg) | Mean | 409.3 | - | - | - |
| MF | 16.16 | - | - | - | |
| 2AA (2 µg) | Mean | - | - | - | 183.3 |
| MF | - | - | - | 21.15 | |
Applicant's summary and conclusion
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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