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EC number: 269-642-9 | CAS number: 68308-30-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test: negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 100, TA 98, E. coli WP2 uvrA
Chromosome aberration: negative in cultured human peripheral lymphocytes and Chinese hamster lung fibroblasts with and without metabolic activation, respectively
Read-across from source substances 2-ethylhexyl oleate (CAS 26399-02-0), oleyl oleate (CAS 3687-45-4) and 2-octyldodecyl isooctadecanoate (CAS 93803-87-3)
Gene mutation in mammalian cells: negative in mouse lymphoma L5178Y cells (TK locus) and Chinese hamster lung fibroblasts (HPRT locus) with and without metabolic activation, respectively
Read-across from source substances 2-ethylhexyl oleate (CAS 26399-02-0) and oleyl oleate (CAS 3687-45-4)
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:
- 09 Oct 2012 - 10 Feb 2013
- 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 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
- Target gene:
- his operon; trp 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 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. - 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. - 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"). - Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Remarks:
- Summary of available data used for the endpoint assessment of the target substance
- Adequacy of study:
- key study
- Justification for type of information:
- refer to analogue justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- 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 applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: Source: CAS 26399-02-0
- Conclusions:
- The available data on suitable source substances 2-ethylhexyl oleate (CAS 26399-02-0), 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) and (Z)-octadec-9-enyl oleate (CAS 3687-45-4) did not show any clastogenic effect in cultured human peripheral lymphocytes or in Chinese hamster fibroblasts (V79). Therefore applying the read-across approach, the target substance Fatty acids, montan-wax, stearyl esters is not predicted to be clastogenic in mammalian cells in vitro.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Remarks:
- Summary of available data used for the endpoint assessment of the target substance
- Adequacy of study:
- key study
- Justification for type of information:
- refer to analogue justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- 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
- Positive controls validity:
- valid
- Remarks on result:
- other: Source: CAS 26399-02-0
- Conclusions:
- The available data on suitable source substances 2-ethylhexyl oleate (CAS 26399-02-0) and (Z)-octadec-9-enyl oleate (CAS 3687-45-4) did not show any mutagenic effect in Chinese hamster lung fibroblasts (V79) and mouse lymphoma L5178Y cells, respectively. Therefore applying the read-across approach, the target substance Fatty acids, montan-wax, stearyl esters is not predicted to be mutagenic in mammalian cells in vitro.
Referenceopen allclose all
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 | |
A further in vitro chromsome aberration test with the source substance 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) is available and revealed no clastogenic properties in cultured human peripheral lymphocytes with and without metabolic activation. The available in vitro chromosome aberration test with the source substance (Z)-octadec-9-enyl oleate (CAS 3687-45-4) revealed no clastogenic properties in Chinese hamster lung fibroblasts (V79) with and without metabolic activation.
A further gene mutation study was available with the source substance (Z)-octadec-9-enyl oleate (CAS 3687-45-4), which did not show any mutagenic effect in Chinese hamster lung fibroblasts (V79) with and without metabolic activation.
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for read-across
Data on genetic toxicity in bacterial cells with Fatty acids, montan-wax, stearyl esters (CAS 68308-30-5) are available, whereas data on in vitro studies on cytogenicity, and in vitro studies on gene mutation in mammalian cells of Fatty acids, montan-wax, stearyl esters (CAS 68308-30-5) were not available.
The assessment was therefore based on studies conducted with source 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 68308-30-5
The mutagenic potential of Fatty acids, montan-wax, stearyl esters (CAS 68308-30-5) was tested in a reverse mutation assay according to OECD 471 under GLP conditions (CiToxLab, 2013). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA were used. Based on the results of a range finding test, tester strains were incubated with the test substance at concentrations of 5, 15.81, 50, 158.1, 500, 1581 snd 5000 µg/plate with and without metabolic activation (phenobarbital and β-naphthoflavone induced rat liver S9-mix) in both experiments. Precipitation was observed at the highest tested concentration of 5000 µg/plate in all tester strains with and without metabolic activation. Vehicle and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. Thus, Fatty acids, montan-wax, stearyl esters (CAS 68308-30-5) did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.
Genetic toxicity (cytogenicity) in mammalian cells in vitro
CAS 26399-02-0
The cytogenetic potential of 2-ethylhexyl oleate (CAS 26399-02-0) was assessed in an in vitro mammalian chromosome aberration test in primary human lymphocytes, performed according to OECD 473 (Notox, 2010). Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). In the first experiment, cells were incubated with test substance concentrations of 3, 10 and 33 µg/mL in ethanol for 3 hours with and without metabolic activation. In the second experiment cells were incubated with 3, 10 and 33 µg/mL for 24 hours followed by 24 hours expression time and 48 hours following 48 hours expression time, all without metabolic activation. 33 µg/mL was chosen as maximum concentration due to limited solubility of the test substance. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level in comparison to the negative controls. The test material demonstrated only modest cytotoxicity. The vehicle (solvent) and positive controls were shown to be 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 3687-45-4
The potential of oleyl oleate (CAS 3687-45-4) to induce chromosomal aberrations was assessed using Chinese hamster V79 cells, in a study performed according to OECD 473 (Cytotest Cell Research, 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 with short-term treatment (4 h) and fixation time 18 and 28 h, without metabolic activation; and with metabolic activation using 18 h treatment time and 18 h fixation time and 28 h treatment time and 28 h fixation time, respectively. The test substance did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, with or without metabolic activation. The mitotic indices of the treated cultures without metabolic activation were 83.4-119% and with metabolic activation 91-127.1%, compared with the vehicle control. Precipitation was observed at concentrations from 100 µg/mL, while no cytotoxicity was noted at any concentration. The vehicle and positive controls were valid.
CAS 93803-87-3
An in vitro chromosomal aberration test was performed with 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) according to OECD 473 (Notox, 1998). Cultured human peripheral lymphocytes were exposed to the test substance at concentrations up to 1000 µg/mL, with and without metabolic activation (S9-mix). In experiment 1, a short-term treatment (3 h) with harvest time 24 and 48 h, was performed without metabolic activation; while the 24 h treatment time with 24 h harvest time and 48 h treatment time with 48 h harvest time, respectively, was done with metabolic activation. In experiment 2, both the 3 h treatment with metabolic activation and the 24 h treatment without metabolic activation had a harvest time of 24 h. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, with or without metabolic activation. The mitotic indices of the treated cultures without metabolic activation were 81-113% and with metabolic activation 68-108%, compared with the vehicle control. No cytotoxicity was noted at any concentrations, but precipitation was observed at concentrations from 1000 µg/mL and above. The vehicle and positive controls were valid.
Genetic toxicity (mutagenicity) in mammalian cells in vitro
CAS 26399-02-0
An in vitro mammalian cell gene mutation assay according to OECD 476 and under GLP was performed with 2-ethylhexyl oleate (CAS 26399-02-0) in mouse lymphoma L5178Y cells (Notox, 2010). Two independent experiments (with 3 or 24 hours of exposure) were performed in the absence and presence of S9-mix with test substance concentrations up to 100 μg/mL dissolved in ethanol. Precipitation was seen at 100 µg/mL and higher. Cyclophosphamide and methylmethanesulfonate were used as positive controls with and without S9-mix, respectively. Positive and negative controls were valid and in range of historical control data. No significant increase in mutation frequency occurred in any of the test conditions, indicating that 2-ethylhexyl oleate is not mutagenic in the mammalian cells in vitro.
CAS 3687-45-4
An in vitro mammalian cell gene mutation assay was performed using oleyl oleate (CAS 3687-45-4), according to OECD 476 (Cytotest Cell Reasearch, 1994). Chinese hamster lung fibroblasts (V79) were treated with oleyl oleate at concentrations of up to 100 µg/mL for 4 h both 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. Both with and without metabolic activation, no increases in mutant frequency were observed in the initial and in the confirmatory gene mutation assay. There was no evidence of excessive cytotoxicity (i.e., < 10 % relative cloning efficiency) at any of the tested concentrations either in the presence or absence of metabolic activation in any of the experiments performed.
Overall conclusion for genetic toxicity
There are data available regarding the mutagenetic potential of the target substance Fatty acids, montan-wax, stearyl esters (CAS 68308-30-5) in bacterial cells. Furthermore, read-across from source substances was applied for in vitro studies on cytogenicity and mutagenicity in mammalian cells. The results of the available studies were consistently negative. Based on the available data and following the analogue approach, no hazard regarding genotoxicity is identified for the target substance Fatty acids, montan-wax, stearyl esters (CAS 68308-30-5).
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 physico-chemical, 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 Fatty acids, montan-wax, stearyl esters (CAS 68308-30-5), data will be generated from data for 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, applying the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.
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