Registration Dossier
Registration Dossier
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EC number: 247-668-1 | CAS number: 26402-26-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
All available in vitro and in vivo genotoxicity studies were found negative, indicating that Fatty Acid Glycerides have no genotoxic potential.
Available in vitro genotoxicity studies on Fatty Acid Glycerides (CAS No.):
- Ames Test: 67701-26-2, 8001-78-3, 91744-13-7, 73398-61-5 and medium and long-chain triglycerides
- Chromosome Aberration: 8001-79-4, 91052-13-0 and medium and long-chain triglycerides
- Mammalian gene mutation test in vitro (HPRT): medium and long-chain triglycerides
- Sister Chromatide Exchange: 8001-79-4
Available in vivo genotoxicity studies on Fatty Acid Glycerides (CAS No.):
- Micronucleus assay: 8001-79-4, 91845-19-1 and medium and long-chain triglycerides
In vitro tests covering genetic toxicity are being performed with the registered substance, octanoic acid, monoester with glycerol. When the new data is available section 7.6 Genetic toxicity and section 2.1 GHS will be updated accordingly.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- migrated information: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Study period:
- 2010-05-03 to 2010-06-12
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- GLP - Guideline study, tested with the source substance Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS 91052-13-0). In accordance to the ECHA guidance document ¿Practical guide 6: How to report read-across and categories (March 2010)¿, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance. Read-across from Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS No. 91052-13-0) for systemic mammalian toxicity endpoints was judged to be justified for the following reasons: This read across substance is also a glyceride, containing mainly C12-14 fatty acid and acetate moiety as well as glycerol. It¿s an organic liquid with a pour point of -8 °C and a melting point of 357.85 °C and a vapour pressure < 0.01 Pa at room temperature. In contrast to the glycerides of the fatty acid glyceride category, it has a higher water solubility of 8.75 mg/L, which might influence its environmental distribution, but not the mammalian metabolism upon systemic uptake. Therefore it is expected to feed into the same mammalian physiological pathways as the members of the fatty acid glyceride category, like citric acid cycle, sugar synthesis and lipid synthesis.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (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:
- other: CHL/IU
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9-mix
- Test concentrations with justification for top dose:
- 6h with and without S9-mix: 0.02, 0.39, 0.078 and 0.156 mg/mL
24 and 48h without S9-mix: 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5.0 mg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor, the test substance was insoluble in water and physiological saline. And the solubility test was performed with DMSO and acetone. The test substance was insoluble at 500.0 mg/mL in DMSO, and was dissolved at 500.0 mg/mL in acetone, and neither generation of gas nor exothermic reaction was observed. Therefore acetone was selected as solvent in this study. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without S9-mix Migrated to IUCLID6: 6h treatment: 0.1 µg/mL, 24+48h treatment 0.05 µg/mL
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- with S9-mix Migrated to IUCLID6: 7 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 6, 24 and 48 hours
- Expression time (cells in growth medium): 18h after 6h treatment
SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid
STAIN (for cytogenetic assays): 0.1% crystal violet solution
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
OTHER EXAMINATIONS:
- Determination of polyploidy: cells carrying greater than 38 chromosomes including triploid were recorded as polyploidy - Evaluation criteria:
- the following criteria were set to evaluate the frequency of aberrant cells in each dose group:
A final judgment was concluded excluding gaps, nevertheless, separate records were kept for both including and excluding gaps.
Negative (-) less than 5%
Equivocal (+-) 5% or more, less than 10%
Positive (+) 10% or more - Statistics:
- No statistical analysis was performed to the results.
- Species / strain:
- other: CHU/IU
- 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
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations of 0.078 µg/mL and higher
RANGE-FINDING/SCREENING STUDIES: In the results of the cell growth inhibition test, the dose of 50% cell growth inhibition was 5.0 mg/mL and
more all of without S9 mix, with S9 mix, 24-hour and 48-hour exposure. In addition, the cell growth inhibition was observed, though the cell growth rate was more than 50% at the 0.156~1.25 mg/mL doses of the 24-hour exposure and the 0,313~1.25 mg/mL doses of 48-hour exposure.
COMPARISON WITH HISTORICAL CONTROL DATA: Yes, in range - Conclusions:
- Interpretation of results: negative
- 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:
- 24 Feb 1994 - 18 Apr 1994
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP-guideline study with acceptable restrictions. No historical control data, insufficient bacteria strain selection, S9-mix from liver induced only with phenobarbiturate
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- yes
- Remarks:
- no historical control data, insufficient bacteria strain selection, S9-mix from liver induced only with phenobarbiturate
- 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:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbiturate-induced rat liver microsomes
- Test concentrations with justification for top dose:
- 8, 40, 200, 1000, 5000 µg/plate
- Vehicle / solvent:
- - Vehicle/solvent used: acetone
- Untreated negative controls:
- yes
- Remarks:
- water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-nitrofluorene (-S9, TA 98 and 1538); 9-aminoacridine (-S9, TA 1537); sodium azide (-S9, TA100 and 1535); aminoanthracene (+S9, all strains).
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Experiment I: main test in medium (February 1994)
Experiment II: preincubation test (April 1994)
DURATION
- Preincubation period: 30 min (only experiment II)
- Exposure duration: 96 h
NUMBER OF REPLICATIONS: triplicates
- 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
- 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
- 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
- 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
- Species / strain:
- S. typhimurium TA 1538
- 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: at 5000 µg/plate
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed. - Conclusions:
- Interpretation of results: negative
- 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:
- 19 - 28 Mar 1996
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Comparable to guideline study with acceptable restrictions. Limited details on test material. The recommended strains TA 102 or E. coli WP2 were not tested. Only one positive control substance was tested in the presence of metabolic activation and not in all strains.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- limited details on test material; the recommended strains TA 102 or E. coli WP2 were not tested; only one positive control substance was tested in the presence of metabolic activation and not in all strains.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (S. typhimurium)
- Species / strain / cell type:
- other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
- Details on mammalian cell type (if applicable):
- - Periodically checked for karyotype stability: yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented mammalian liver post-mitochondrial fraction (S-9 mix), prepared from the livers of male Sprague Dawley rats induced with Aroclor 1254 (intraperitoneal injection of 500 mg/mL for 5 consecutive days).
- Test concentrations with justification for top dose:
- 0.1 mL/plate of 10% (v/v) test solution, with or without metabolic activation
- Vehicle / solvent:
- - Vehicle/solvent used: DMSO (10 µL/plate)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Dexon (4-[(Dimethylamino)phenyl]diazene sulfonic acid, sodium salt) (-S9 and +S9, TA 98, TA 100 and TA 1537); 2-nitrofluorene (-S9 and +S9, TA 1538); 2-aminofluorene (-S9 and +S9, TA 100 and TA 1538); sodium azide (S9 and +S9, TA 1535)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION 1: spot plate technique as preliminary toxicity screen
METHOD OF APPLICATION 2: in agar (plate incorporation) for the main experiment
DURATION
- Exposure duration: 48-72 h
NUMBER OF REPLICATIONS: triplicate each
DETERMINATION OF CYTOTOXICITY
- Method: assessment of growth inhibition using the spot plate technique - Evaluation criteria:
- The test substance was considered "potentially mutagenic", if a two-fold or greater increase in the mean number of spontaneous revertants was observed in treated cultures compared to control cultures.
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Positive controls in the presence of metabolic activation: only one positive control substance was tested in the presence of metabolic activation and not in all strains (only in TA 100 and TA 1535). However, the proper activity of the S9-mix was demonstrated in TA 1537 using 2-aminofluorene as positive control which requires metabolic activation.
RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity screening with 10% (v/v) test substance solution in DMSO was carried out in all strains of S. typhimurium. Using the spot plate technique, the test substance was applied on a sterile filter paper, which was placed on the solidified agar containing the bacterial cultures. Following treatment for 24-48 h, no growth inhibition was observed in the area of the filter paper. Dexon was used as positive control and caused a positive zone of growth inhibition after application to the culture plates. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- migrated information: read-across based on grouping of substances (category approach)
- Adequacy of study:
- weight of evidence
- Study period:
- 17 Apr -21 Apr 1997
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP-Guideline study with acceptable restrictions. Only 4 S. typhimurium strains used instead of 5 as required according to the current criteria. Only one concentration (5000 µg/plate) tested due to insolubility of the test substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in 1983
- Deviations:
- yes
- Remarks:
- only 4 S. typhimurium strains used instead of 5 as required according to the current criteria. Only one concentration (5000 µg/plate) tested due to insolubility of the test substance.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Düsseldorf, Germany
- 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
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital/β-Naphthoflavone
- Test concentrations with justification for top dose:
- 5000 µg/plate with and without metabolic activation
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: 95% ethanol (100 and 50 µL/plate in the plate incorporation and preincubation tests, respectively).
- Justification for choice of solvent/vehicle: in a preliminary test, the solubility of the test compound was determined in a number of solvents suitable for the Ames test, namely water, dimethylsulfoxide, glycerol, dimethyl formamide, formamide, ethanol, acetone, dioxane, tetrahydrofuran and tetrahydrofurfuryl alcohol. In all these solvents, the test compound could not be dissolved in appreciable amounts. Therefore, a suspension of the test material was prepared on the day of testing by mixing the test compound with 95% ethanol, stirring it on a magnetic stirrer and using samples of this stirred suspension for testing. No stability testing or composition analysis was performed on the dosing suspension. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: -S9: 2-nitrofluorene (2-NF; 2.5 µg/plate for TA98); sodium azide (SA; 5 and 2.5 µg/plate for TA100 and TA1535, respectively); 9-aminoacridine (9-AA; 40 µg/plate for TA1537; +S9: 2-aminoanthracene (2-AA; 2.5 µg/plate for all strains)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
First experiment: in agar (plate incorporation)
Second experiment: preincubation
DURATION
- Preincubation period: 30 min (second experiment)
- Exposure duration: 72 h (first and second experiment)
NUMBER OF REPLICATIONS: 3 replications in the first and second experiment, respectively.
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth - Evaluation criteria:
- Criteria for determination of a valid test:
The following criteria had to be met for the mutagenicity assay to be considered valid:
- In the solvent control, each tester strain culture exhibited a characteristic mean number of spontaneous revertants.
- To ensure that appropriate numbers of bacteria were plated, overnight culture titers had to be in excess of 1E08 bacteria/mL.
- The mean of each positive control exhibited a significant increase in the number of revertants over the mean value of the respective vehicle control.
- In a standard Ames test, at least four non-toxic dose levels were required to evaluate the assay data. In the current test, due to non-solubility of the test compound, only a limit dose was employed.
Criteria for evaluation of test results:
For a test compound to be considered positive, it had to (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. In a standard Ames test, this increase had to be accompanied by a dose response towards increasing concentrations of the test article. In the current test, a reproducible caused by a treatment with the limit dose would have been sufficient. A test article that did not meet these criteria would be called non-mutagenic in bacteria. Single increases in revertant frequencies, which were not reproducible in two independent tests were considered non-relevant. - Statistics:
- Mean values and standard deviation were calculated.
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- due to insolubility, the test mateial was tested as suspension at a single limit concentration of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- due to insolubility, the test mateial was tested as suspension at a single limit concentration of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- due to insolubility, the test mateial was tested as suspension at a single limit concentration of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- due to insolubility, the test mateial was tested as suspension at a single limit concentration of 5000 µg/plate
- 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
- Water solubility: the test substance is not soluble in water.
- Precipitation: due to non-solubility of the test susbtance in all solvents used routinely in the Ames test, the tester strains were exposed to a suspension of the test compound at a single limit concentration of 5000 µg/plate. Test compound-treated bacterial colonies were counted by hand, as the precipitate formed by the test compound prevented automatic counting. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- migrated information: read-across based on grouping of substances (category approach)
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI medium supplemented with 10% horse serum, 200 µg/mL sodium pyruvate and 50 µg/mL gentamycin
- Properly maintained: yes (stock cultures were kept in a liquid nitrogen tank to start new stock cultures periodically in which cells were diluted daily and kept at a density of about 2E+5 to 1.5E+6)
- Periodically checked for Mycoplasma contamination: yes - 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 (500 mg/kg bw)
- Test concentrations with justification for top dose:
- First experiment:
with and without metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL
Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 3600 µg/mL
with metabolic activation: 156, 313, 625, 1250, 2500 and 3600 µg/mL
Repeat of second test:
without metabolic activation: 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: N-ethyl-N-nitrosourea (50 µg/mL, -S9), 7,12-dimethyl-1,2-benzanthracene (3.3 µg/mL, +S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
First test:
- Exposure duration: +S9: 3 h, -S9: 4 h
Second main test:
- Exposure duration: +S9: 3 h, -S9: 24 h
Repeat of second main test:
- Exposure duration: -S9: 24 h
(In cell cultures with metabolic activation, the treatment period was limited to 3 h due to the cytotoxic effects induced by S9.)
- Expression time (cells in growth medium): 3 days (counting from the start of the experiment), cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates. The cell density was counted and adjusted to 3E+5 cells/mL daily.
- Selection time: approx. 10 days, cells were seeded in 2 microtitre plates with a density of 2000 cells/well in TFT selctive medium to determine the number of mutants.
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 14 days
SELECTION AGENT (mutation assays): trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: duplicates
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (cytotoxicity corresponds to relative survival compared to the respective negative control values)
OTHER EXAMINATIONS:
Cloning efficiency was determined by seeding exposed cells in one microtiter plate with a density of 2 cells/well in medium without TFT.
Small and large colonies were differentiated as small colonies are capable to indicate chromosomal mutations. - Evaluation criteria:
- The test item was considered as mutagenic when all of the following criteria were met:
- increases in the mutation frequency were observed in treated cultures compared to the corresponding negative control values at one or more test concentrations
- the increases showed a dose-response relationship
- the increases were reproducible between the replicates and the first and second test (when treatment conditions were the same)
- the increases were statistically significant
- the increases exceeded the historical negative control range
- the relative survival of the test groups was at least 15% at the end of the treatment period
When the above mentioned criteria were not met, the test item was considered as non-mutagenic. - Statistics:
- Single values of the duplicates were determined from the examined parameters. Statistical analyses were performed with the SAS (R) procedures version 8.1 (SAS Institute Inc., Cary, North Carolina 27513, USA). In detail, mutation frequencies of treated samples were compared to the correpsonding negative controls with the analyis of variance test.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- without S9: starting from 3600 µg/mL (first test (4 h exposure)) and 160 µg/mL (second test (24 h exposure)), with S9: starting from 2500 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the negative and positive controls were all within the range of the historical control data despite one positive control value which was slightly higher that the historical control value. Thus, the study was considered to be valid. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- migrated information: read-across based on grouping of substances (category approach)
- Adequacy of study:
- weight of evidence
- Study period:
- 16 Oct - 18 Apr 2002
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (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:
- primary culture, other: human lymphocytes
- 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 (500 mg/kg bw)
- Test concentrations with justification for top dose:
- Preliminary toxicity test:
with and without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL
First experiment (and repeat tests):
without metabolic activation: 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL
Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL
without metabolic activation (repeat): 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL
Only slides from cultures of the following dose groups were selected for metaphase analyses:
First experiment:
without metabolic activation: 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL
Second experiment:
without metabolic activation (repeat): 40, 80 and 160 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: daunomycin (0.015 µg/mL, -S9), cyclophosphamide (6 µg/mL, +S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
Preliminary test and first main test:
- Exposure duration: 3 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)
Second main test:
- Exposure duration: +S9: 3 h, -S9: 20 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)
SPINDLE INHIBITOR (cytogenetic assays): demecolcine (0.1 µg/mL)
STAIN (for cytogenetic assays): 3% Giemsa
NUMBER OF REPLICATIONS: duplicates
NUMBER OF CELLS EVALUATED: at least 100 metaphases (when possible) and 1000 cells for the determination of mitotic index
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (calculated as percentage of cells in metaphases)
OTHER EXAMINATIONS:
- Determination of polyploidy: yes, defined as metaphases with multiples of the haploid chromosome number other than diploid (e.g. 3n, 4n etc.) and determined in 200 metaphases
- Determination of endoreplication: yes, defined by the presence of chromosomes with 4, 8 chromatids and determined in 200 metaphases - Evaluation criteria:
- EVALUATION OF RESULTS
The study was considered as valid when:
- the negative control cultures showed a low frequency of metaphases with chromosome aberrations, normally 0 - 3% (excluding gaps)
- the positive control cultures showed a clear increase in the frequency of metaphases with chromosome aberrations
For the evaluation of the results, the number of metaphases with chromosome aberrations of each test condition were compared to the concurrent negative control. Gaps were recorded but excluded from the analyses.
The test material was considered clastogenic in this test system if all of the following criteria were met:
1. increases in the frequency of chromosome aberrations were determined at one or more test concentrations
2. reproducible increases in aberrant chromosomes between replicates
3. statistically significance in the increases of chromosome aberrations
4. increases exceed the historical negative control range
5. increases were not associated with large changes in pH or osmolarity
The evidence of a dose-response relation ship was considered to support the conclusion.
The test material was considered non-clastogenic in this test system when the increase in chromosomal aberrations was not statistically significanct and/or no reproducibility was observed.
Results which failed to meet the above mentioned criteria were considered as equivocal. - Statistics:
- When appropriate, Fischer´s Exact Test was performed to evaluate statistical significance.
- Species / strain:
- primary culture, other: human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- dose-related toxicity which reduced the mitotic index in the high-dose group (5000 µg/mL) to 64% of the vehicle control without metabolic activation and to 10% with metabolic activation (table 1)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- First main test
As the frequencies of metaphases with chromosomal aberrations were in general unacceptably high (4% for the duplicates without metabolic activation and 7 or 3% for the duplicates with metabolic activation), the repetition of the first experiment was conducted with the identical experimental design as the initial test. The values for chromosome aberrations within the test samples were between 1 and 9%, but they were not reproducible between the replicates nor did they show any dose-related effect (the data from the initial experiment are not included in the study report).
Scoring of slides prepared from the repeat of the initial experiment revealed no appropriate increases in chromosomal aberrations within the positive control samples without metabolic activation. Thus, this part of the test was considered as invalid and therefore repeated.
Second main test
As the samples without metabolic activation revealed mean mitotic indices lower than 50% of the solvent control in all dose groups (data not shown), this part of the test was repeated with lower dosages in the second test (table 3).
Polyploid and endoreduplicated metaphases
Single polyploid metaphases were observed at few test points without showing a dose-relation ship. Therefore, this effect is considered as incidental and not treatment-related. In contrast, no endoreduplicated metaphases were observed.
In each test group despite the two positive controls treated with cyclophosphamide, 100 metaphases were counted. In the cyclophosphamide treated samples only 59 and 30 scorable metaphases were detected.
Test validity
The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values (table 4). The positive controls produced statistically significant increases in the frequency of metaphases with chromosomal aberrations in the valid parts of the tests, thereby demonstrating the sensitivity of the test and the efficacy of the S9 mix. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- migrated information: read-across based on grouping of substances (category approach)
- Adequacy of study:
- weight of evidence
- Study period:
- 03 May 2010 - 12 Jun 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (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:
- other: CHL/IU
- 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 5,6-benzoflavone
- Test concentrations with justification for top dose:
- 6h treatment with and without S9-mix: 0.02, 0.39, 0.078 and 0.156 mg/mL
24 and 48h treatment without S9-mix: 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5.0 mg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor, the test substance was insoluble in water and physiological saline. And the solubility test was performed with DMSO and acetone. The test substance was insoluble at 500.0 mg/mL in DMSO, and was dissolved at 500.0 mg/mL in acetone, and neither generation of gas nor exothermic reaction was observed. Therefore acetone was selected as solvent in this study. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without S9-mix Migrated to IUCLID6: 0.1 µg/mL ( 6 h treatment), 0.05 µg/mL (24 and 48 h treatment)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- with S9-mix Migrated to IUCLID6: 7 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 6, 24 and 48 h
- Expression time (cells in growth medium): 18 h after 6 h treatment
SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid
STAIN (for cytogenetic assays): 0.1% crystal violet solution
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
OTHER EXAMINATIONS:
- Determination of polyploidy: yes, cells carrying greater than 38 chromosomes including triploid were recorded as polyploidy - Evaluation criteria:
- The following criteria were set to evaluate the frequency of aberrant cells in each dose group:
A final judgment was concluded excluding gaps, nevertheless, separate records were kept for both including and excluding gaps.
Negative (-) less than 5%
Equivocal (+-) 5% or more, less than 10%
Positive (+) 10% or more - Species / strain:
- other: CHU/IU
- 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
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations of 0.078 µg/mL and higher
RANGE-FINDING/SCREENING STUDIES: In the results of the cell growth inhibition test, the dose of 50% cell growth inhibition was 5.0 mg/mL and
more all of without S9 mix, with S9 mix, 24 h and 48 h exposure. In addition, the cell growth inhibition was observed, though the cell growth rate was more than 50% at the 0.156~1.25 mg/mL doses of the 24 h exposure and the 0,313~1.25 mg/mL doses of 48 h exposure.
COMPARISON WITH HISTORICAL CONTROL DATA: Yes - Remarks on result:
- other: strain/cell type:
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- migrated information: read-across based on grouping of substances (category approach)
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study with acceptable restrictions. Report does not contain all study details.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- lack of cytotoxicity data
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable.
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- no data
- 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:
- 1600, 3000, 5000 µg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- Migrated to IUCLID6: mitomycin C (-9); cyclophosphamide (+S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 10 h without S9, 2 h with S9
SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa
Cells were arrested in first metaphase by addition of colcemid and harvested by mitotic shake off, fixed, and stained in 6% Giemsa.
In the absence of S9, cells were incubated with test compound or solvent for 10 h at 37°C. Cells were then washed and fresh medium containing colcemid was added for an additional 3 h followed by harvest.
In the presence of S9, cells were incubated with test compound or solvent for 2 h at 37°C. Cells were then washed, medium without test compound was added, and incubation was continued for 10 h. Colcemid was added for the last 3 h of incubation before harvest. S9 was from the livers of Aroclor 1254-induced male Sprague Dawley rats. - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- .
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- migrated information: read-across based on grouping of substances (category approach)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Acceptable, well-documented publication meeting basic scientific principles. Test compound was a mixture of short- and long-chain acyl triglyceride.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- , only 100 cells per dose scored.
- Principles of method if other than guideline:
- The SALATRIM family of triacylglycerols differs from other fats in the ratio of short-chain fatty acids (SCFA) to long-chain fatty acids (LCFA) and in that stearic acid is the major LCFA. These fats have caloric availability values (4.5-6 kcal/g) lower than that of corn oil (9 kcal/g). SALATRIM 23CA Lot
A014, a typical SALATRIM fat, was tested in in vitro mammalian cell genotoxicity assays including the chromosomal aberration, unscheduled DNA synthesis, and HPRT mammalian cell mutagenesis assays. Corn oil also was tested as a reference fat. Both the SALATRIM fat and corn oil were negative
in the three assays. - GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- Chinese hamster ovary cells (CHO ETCC CCL 61 CHO-KL, proline-requiring)
- Type and identity of media: Cells were grown in a 5% C02 atmosphere at 37 "C in McCoy's 5a medium. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- 0, 250, 500, 1000 µg/mL.
The high dose was limited by the low solubility of the fats in the assay medium. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
Acetone was used as the solvent for both SALATRIM 23CA lot A014 and corn oil. - Untreated negative controls:
- yes
- Remarks:
- Corn oil doses were 500, 750 and 1000 µg/mL
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: methylmethanesulfonate (-S9) and cyclophosphamide (+S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium;
DURATION
- Exposure duration: 8h (-S9); 2h (+S9);
In the absence of metabolic activation, the fats and 0.01 mM deoxybromouridine (BrdU) were added to the cell culture and incubated at 37ºC for 8h. The cells were washed and then incubated in fresh medium containing 0.01 mM BrdU for 13.5 h.
When metabolic activation was used, the cultures were exposed to the fats for 2 h at 37ºC, washed, and then incubated in fresh medium containing
0.01 mM BrdU for 6-8 h.
After incubation in BrdU, colchicine was added to the cultures at 0.4 ¿g/mL followed by incubation for 2.5 h at 37 ºC.
SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.4 µg/mL
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: duplicates
NUMBER OF CELLS EVALUATED: 50 cells per flask were evaluated for chromosomal aberrations, resulting in a total of 100 cells evaluated per dose.
DETERMINATION OF CYTOTOXICITY
- Method: The mitotic index was evaluated on the basis of at least 1000 cells per flask. - Evaluation criteria:
- A test material would be considered positive if there was a statistically significant (p < 0.05) increase in the frequency of cells with chromosomal damage and if this increase was dose-dependent.
- Statistics:
- The number of cells with chromosomal damage in the test fat groups and positive control group were compared to the concurrent solvent control by Fischer's exact test (Gad and Weil, 1991).
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The SALATRIM fat appeared to be soluble in the culture medium for the chromosomal aberration assay at a concentration of 40 ¿g/mL but produced a suspension at higher concentrations.
The pH of the high dose in the medium was approximately 7.0. The preliminary dose range/cytotoxicity study used a dose range of 0-1000 ¿g/mL and was limited by the lack of solubility of the fat. Corn oil behaved in a manner similar to that of SALATRIM 23CA lot A014 and was tested using the same criteria. No significant cell cycle delay or reduction in the mitotic index was noted with either corn oil or the SALATRIM fat. There was a slight increase in the number of cells in metaphase 1 at a SALATRIM dose of 1000 ¿g/mL and at corn oil doses of 40 and 200 ¿g/mL, but these were not significant on the basis of assay criteria. Therefore, for the definitive assay, a dose range of 0 (solvent control), 250, 500, and 1000 ¿g/mL was chosen for the SALATRIM and 500,750, and 1000 ¿g/mL for corn oil.
RANGE-FINDING/SCREENING STUDIES:
A preliminary dose range study to determine cytotoxicity and optimal cell fixation time was done before the definitive study was performed. Doses of 8.0, 40.0, 200.0, 500.0, and 1000 ¿g/mL were tested.
On the basis of the results of the range-finding study, doses of 250,500, and 1000 ¿g/mL for the SALATRIM fat and 500,750, and 1000 ¿g/mL for corn oil were used for the definitive study and a harvest time of 8-10 h was selected.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of metabolic activation, no major changes were noted in the mitotic index with either SALATRIM 23CA lot A014 or corn oil, indicating a lack of cytotoxicity. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- migrated information: read-across based on grouping of substances (category approach)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Acceptable, well-documented publication meeting basic scientific principles. Test compound was a mixture of short- and long-chain acyl triglyceride.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- exposure duration not reported;
- Principles of method if other than guideline:
- The SALATRIM family of triacylglycerols differs from other fats in the ratio of short-chain fatty acids (SCFA) to long-chain fatty acids (LCFA) and in that stearic acid is the major LCFA. These fats have caloric availability values (4.5-6 kcal/g) lower than that of corn oil (9 kcal/g). SALATRIM 23CA Lot
A014, a typical SALATRIM fat, was tested in in vitro mammalian cell genotoxicity assays including the chromosomal aberration, unscheduled DNA synthesis, and HPRT mammalian cell mutagenesis assays. Corn oil also was tested as a reference fat. Both the SALATRIM fat and corn oil were negative
in the three assays. - GLP compliance:
- not specified
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- Chinese hamster ovary (CHO) CHO-K1 cells obtained from the American Type Culture Collection
- Type and identity of media: in Ham¿s F12 medium with 31 µg/mL penicillin, 50 µg/mL streptomycin sulfate, and 5 % heatinactivated
fetal bovine serum (F12/5). - Metabolic activation:
- with and without
- Metabolic activation system:
- final S-9 concentration of 1 %
- Test concentrations with justification for top dose:
- 0, 31.25, 62.5, 125, 250, 500 and 1000 ¿g/mL
The high dose was limited by the low solubility of the fats in the assay medium. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
Stock solutions of SALATRIM 23CA lot A014 and corn oil were made in acetone such that the acetone never exceeded 1 % of the culture. - Untreated negative controls:
- yes
- Remarks:
- Corn oil doses were 327.7-1000 ¿g/mL.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Positive controls were EMS (200 ¿g/mL of culture) without metabolic activation and 3-MC (5 ¿g/mL of culture) with metabolic activation.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium;
DURATION
- Exposure duration: not reported
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14-17 days
Phenotypic expression of induced mutants was conducted by incubating the cells for 7 days. Cells were subcultured every 2-3 days by trypsinizing the flask, counting the cells, and replating 2 x 10(6) cells. After the expression period, approximately 3 x 10(6) cells from each culture were seeded in 100 mL of cloning medium supplemented with TG for selection of resistant cells. Approximately 600 cells were seeded in 100 mL of TG-free medium to determine the percentage of viable cells. The cells were incubated for 14-17 days and cell colonies counted with an ARTEK colony counter. Mutant frequency (ratio of mutant cells to nonmutant cells) was calculated by dividing the number of resistant colonies by the number of unselected viable colonies.
SELECTION AGENT (mutation assays): The selective cloning medium contained 30 µM 6-thioguanine (TG).
NUMBER OF REPLICATIONS: The definitive study was done in duplicate using duplicate cultures for each replicate.
DETERMINATION OF CYTOTOXICITY
Cytotoxicity was determined by detaching the cells from the culture flask with 0.05% trypsin-0.02% EDTA. A Model 2F Coulter counter was used to determine cell numbers, and an aliquot containing at least 1-2 x 10(6) cells was added to 20 mL of F12/5 to determine phenotypic expression. The remaining cell suspension was diluted in approximately 35 mL of medium so that 500 cells were plated into two Petri dishes. After incubation for 14-17 days, cell colonies were determined. Survival was expressed as the cloning efficiency (CE) relative to the solvent control. - Evaluation criteria:
- The test results were considered positive if a dose-related increase in the number of mutant colonies occurred and the mutant frequencies of duplicate cultures treated with one or more concentrations of the test article were at least 3 times the average of those from the solvent control cultures.
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Although the test material was soluble in the acetone, turbidity was noted in the cultures, indicating solubility had been exceeded.
RANGE-FINDING/SCREENING STUDIES:
Preliminary experiments were done to determine cytotoxicity and aid in selection of the dose range. - Conclusions:
- Interpretation of results: negative
SALATRIM 23CA lot A014 presented no evidence of mutagenic potential under the conditions of the assay.
Positive controls gave the expected results, validating the assay.
Corn oil did not alter either the relative cloning efficiency or the mutation frequency of the cells.
Referenceopen allclose all
Table 1: Effects of the test substance on viability and chromosome aberrations
Test item |
Concentration |
Cell viability |
Aberrant cells in % |
|
|
in µg/mL |
in % |
Numerical |
Structural including gaps |
Exposure period 6 hrs without S9 mix |
||||
Untreated |
-- |
-- |
0.0 |
0.0 |
Solvent |
-- |
100 |
0.0 |
1.5 |
MMC |
0.010 |
105.5 |
0.0 |
39.5 |
Test substance |
0.020 |
101.0 |
0.5 |
0.0 |
0.039 |
98.0 |
1.0 |
1.5 |
|
0.078 |
88.0 |
0.0 |
0.0 |
|
0.156 |
82.5 |
0.5 |
1.0 |
|
Exposure period 6 hrs with S9 mix |
||||
Untreated |
-- |
-- |
0.0 |
2.0 |
Solvent |
-- |
100 |
0.0 |
0.5 |
B(a)P |
7.000 |
96.5 |
0.0 |
28.5 |
Test substance |
0.020 |
96.0 |
0.0 |
2.0 |
0.039 |
92.0 |
0.0 |
0.0 |
|
0.078 |
86.0 |
1.0 |
0.5 |
|
0.156 |
84.5 |
0.5 |
1.0 |
|
Exposure period 24 hrs without S9 mix |
||||
Untreated |
-- |
-- |
1.0 |
0.5 |
Solvent |
-- |
100 |
0.5 |
0.0 |
MMC |
0.050 |
102.0 |
0.0 |
41.5 |
Test substance |
0.078 |
90.5 |
0.0 |
0.5 |
0.156 |
82.5 |
0.0 |
1.0 |
|
0.313 |
76.0 |
1.0 |
1.0 |
|
0.625 |
66.0 |
0.0 |
0.5 |
|
1.250 |
66.0 |
0.5 |
2.0 |
|
2.500 |
84.5 |
0.5 |
0.0 |
|
5.000 |
92.0 |
0.0 |
1.0 |
|
Exposure period 48 hrs without S9 mix |
||||
Untreated |
-- |
-- |
0.0 |
0.0 |
Solvent |
-- |
100 |
0.5 |
0.0 |
MMC |
0.050 |
94.0 |
0.0 |
58.5 |
Test substance |
0.078 |
99.0 |
0.0 |
1.0 |
0.156 |
86.5 |
0.0 |
0.5 |
|
0.313 |
74.5 |
1.0 |
0.0 |
|
0.625 |
70.0 |
0.0 |
0.0 |
|
1.250 |
79.5 |
0.5 |
0.0 |
|
2.500 |
87.5 |
0.5 |
0.0 |
|
5.000 |
92.5 |
0.5 |
1.0 |
|
MMC: Mitomycin C
B(a)P: Benz(a)pyrene
Table 1: Ames Test Results - main test
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 1535 |
TA 1538 |
TA 100 |
TA98 |
TA1537 |
||
- |
Negative control (water) |
14 |
22 |
146 |
34 |
21 |
- |
0 (Solvent control) |
12 |
25 |
154 |
38 |
18 |
- |
8 |
13 |
21 |
172 |
37 |
26 |
- |
40 |
9 |
30 |
162 |
36 |
19 |
- |
200 |
9 |
30 |
133 |
32 |
25 |
- |
1000 |
12 |
33 |
136 |
40 |
21 |
- |
5000 |
11P |
31P |
149P |
34P |
25P |
Positive controls - S9 |
Name |
SA |
NF |
SA |
NF |
9AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
2.5 |
2.5 |
25 |
|
Number of colonies/plate |
354 |
108 |
376 |
101 |
105 |
|
+ |
Negative control (water) |
16 |
29 |
179 |
65 |
19 |
+ |
0 (Solvent control) |
16 |
36 |
145 |
59 |
21 |
+ |
8 |
20 |
38 |
168 |
46 |
17 |
+ |
40 |
19 |
43 |
173 |
52 |
31 |
+ |
200 |
23 |
34 |
175 |
65 |
28 |
+ |
1000 |
20 |
30 |
173 |
54 |
23 |
+ |
5000 |
18P |
31P |
170P |
49P |
23P |
Positive controls + S9 |
Name |
AA |
AA |
AA |
AA |
AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
2.5 |
2.5 |
2.5 |
|
Number of colonies/plate |
95 |
458 |
1074 |
690 |
113 |
|
P = Precipitation
SA = Sodium azide
NF = Nitrofluorene
AA = Aminoanthracene
9AA = 9-aminoacridine
P = Precipitate
Table 2: Ames Test Results - preincubation test
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 1535 |
TA 1538 |
TA 100 |
TA98 |
TA1537 |
||
- |
Negative control (water) |
13 |
20 |
169 |
32 |
18 |
- |
0 (Solvent control) |
14 |
21 |
165 |
36 |
21 |
- |
8 |
14 |
20 |
163 |
40 |
23 |
- |
40 |
14 |
23 |
176 |
33 |
15 |
- |
200 |
16 |
24 |
161 |
39 |
23 |
- |
1000 |
12 |
25 |
169 |
39 |
20 |
- |
5000 |
15P |
23P |
167P |
33P |
18P |
Positive controls - S9 |
Name |
SA |
NF |
SA |
NF |
9AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
2.5 |
2.5 |
25 |
|
Number of colonies/plate |
350 |
145 |
401 |
110 |
73 |
|
+ |
Negative control (water) |
17 |
33 |
154 |
37 |
13 |
+ |
0 (Solvent control) |
21 |
39 |
158 |
43 |
16 |
+ |
8 |
15 |
34 |
166 |
41 |
16 |
+ |
40 |
14 |
38 |
165 |
41 |
16 |
+ |
200 |
17 |
39 |
161 |
47 |
13 |
+ |
1000 |
15 |
35 |
161 |
41 |
17 |
+ |
5000 |
17P |
29P |
162P |
44P |
18P |
Positive controls + S9 |
Name |
AA |
AA |
AA |
AA |
AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
2.5 |
2.5 |
2.5 |
|
Number of colonies/plate |
174 |
665 |
1439 |
1270 |
174 |
|
P = Precipitation
SA = Sodium azide
NF = Nitrofluorene
AA = Aminoanthracene
9AA = 9-aminoacridine
P = Precipitate
Table1. Test results of experiment (plate incorporation)
Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD) |
|||||
EXPERIMENT (plate incorporation) |
|||||
S9-Mix |
Without
|
||||
Concentration (per plate) |
TA 98 |
TA100 |
TA1535* |
TA1537* |
TA 1538* |
SC |
40 ± 14 |
163 ± 12 |
10 ± 1 |
12 ± 1 |
9 ± 1 |
Test material |
|
|
|
|
|
0.1 mL |
30 ± 8 |
136 ± 14 |
10 ± 2 |
10 ± 1 |
9 ± 2 |
PC |
|
|
|
|
|
Dexon |
1355 ± 321 |
1397 ± 281 |
- |
432 ± 89 |
- |
NaN3 |
- |
- |
1403 ± 282 |
- |
- |
2-NF |
- |
- |
- |
- |
1253 ± 91 |
2-AF |
- |
188 ± 9 |
- |
- |
8 ± 2 |
S9-Mix
|
With |
||||
Concentration (per plate) |
TA 1535 |
TA1537 |
TA98 |
TA100 |
E.coli WP2 uvrA |
NC |
28 ± 7 |
123 ± 6 |
10 ± 1 |
9 ± 1 |
10 ± 1 |
Test material |
|
|
|
|
|
0.1 mL |
24 ± 2 |
121 ± 2 |
10 ± 1 |
9 ± 3 |
8 ± 1 |
PC |
|
|
|
|
|
Dexon |
1061 ± 197 |
1344 ± 194 |
- |
363 ± 65 |
- |
NaN3 |
- |
- |
1355 ± 337 |
- |
- |
2-NF |
- |
- |
- |
- |
1499 ± 155 |
2-AF |
- |
1205 ± 61 |
- |
- |
1157 ± 218 |
NC = Negative Control; SC = Solvent control; PC = Positive control substances; SD = standard deviation; NaN3 = sodium azide; 2-NF = 2-nitrofluorene; 2-AF = 2-aminofluorene * Tester strains TA 1535 and TA 1537 were found to be contaminated (colonies too numerous to count) and were repeated. Tester strain TA 1538 was found to be out of the expected spontaneous revertant rate and was repeated. Values presented for tester strains TA 1535, TA 1537 and TA 1538 were those of the retest. In the tester strain TA 1538, the positive control substance 2-AF verified the activity of the S-9 mix |
|||||
Table 1. Test Results of Experiment 1 (plate incorporation).
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
|||
Base-pair substitution type |
Frameshift type |
||||
TA 100 |
TA1535 |
TA98 |
TA1537 |
||
– |
untreated |
110 |
8 |
31 |
12 |
– |
Ethanol |
105 |
7 |
33 |
16 |
– |
5000 |
125P |
3P |
30P |
20P |
Positive controls, –S9 |
Name |
SA |
SA |
2-NF |
9-AA |
Concentrations (μg/plate) |
5 |
2.5 |
2.5 |
40 |
|
Mean No. of colonies/plate (average of 3) |
575 |
267 |
123 |
125 |
|
+ |
untreated |
126 |
11 |
25 |
12 |
+ |
Ethanol |
116 |
14 |
33 |
16 |
+ |
5000 |
136P |
8P |
29P |
10P |
Positive controls, + S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
2.5 |
2.5 |
|
Mean No. of colonies/plate (average of 3) |
1394 |
123 |
835 |
120 |
|
SA = sodium azide
2-NF = 2-nitrofluorene
9-AA = 9-aminoacridine
2-AA = 2-Aminoanthracene
P = Precipitate
Table 2. Test Results of Experiment 2 (preincubation).
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
|||
Base-pair substitution type |
Frameshift type |
||||
TA 100 |
TA1535 |
TA98 |
TA1537 |
||
– |
untreated |
142 |
13 |
30 |
14 |
– |
Ethanol |
126 |
9 |
28 |
11 |
– |
5000 |
127P |
8P |
24P |
12P |
Positive controls, –S9 |
Name |
SA |
SA |
2-NF |
9-AA |
Concentrations (μg/plate) |
5 |
2.5 |
2.5 |
40 |
|
Mean No. of colonies/plate (average of 3) |
575 |
267 |
123 |
125 |
|
+ |
untreated |
134 |
7 |
26 |
13 |
+ |
Ethanol |
155 |
11 |
27 |
10 |
+ |
5000 |
128P |
10P |
31P |
13P |
Positive controls, + S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
2.5 |
2.5 |
|
Mean No. of colonies/plate (average of 3) |
839 |
126 |
657 |
76 |
|
SA = sodium azide
2-NF = 2-nitrofluorene
9-AA = 9-aminoacridine
2-AA = 2-Aminoanthracene
P = Precipitate
Table 1: First Experiment - 4 h exposure - Without Metabolic Activation |
|
|
||||
Concentration (µg/mL) |
Cloning efficiency (%) |
Relative Total Growth (%) |
Mutants per 1E+4 surviving cells |
Mutation factor |
|
|
Day 0 |
Day 3 |
|
||||
0 (DMSO) |
100 |
100 |
100 |
2.36 |
1 |
|
625 |
91 |
116 |
57 |
2.04 |
0.86 |
|
1250 |
108 |
94 |
65 |
2.44 |
1.03 |
|
2500 |
106 |
83 |
36 |
2.66 |
1.12 |
|
3600 |
89 |
76 |
11 |
0.30* |
0.12 |
|
5000 |
74 |
83 |
8 |
0.04* |
0.017 |
|
ENU (50) |
53 |
62 |
45 |
# |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 2: First Experiment - 3 h exposure - With Metabolic Activation |
|
|
||||
Concentration (µg/mL) |
Cloning efficiency (%) |
Relative Total Growth (%) |
Mutants per 1E+4 surviving cells |
Mutation factor |
|
|
Day 0 |
Day 3 |
|
||||
0 (DMSO) |
100 |
100 |
100 |
2.39 |
1 |
|
625 |
75 |
81 |
65 |
2.59 |
1.08 |
|
1250 |
92 |
44 |
46 |
3.04 |
1.27 |
|
2500 |
93 |
30 |
9 |
0.9 |
0.38 |
|
3600 |
90 |
5 |
1 |
0.2 |
0.08 |
|
5000 |
84 |
4 |
1 |
0.47 |
0.2 |
|
DMBA (3.3) |
24 |
50 |
29 |
# |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 3: Second Experiment - 24 h exposure - Without Metabolic Activation |
|
|||||
Concentration (µg/mL) |
Cloning efficiency (%) |
Relative Total Growth (%) |
Mutants per 1E+4 surviving cells |
Mutation factor |
|
|
Day 0 |
Day 3 |
|
||||
0 (DMSO) |
100 |
100 |
100 |
not reported |
not reported |
|
313 |
82 |
9 |
2 |
not reported |
not reported |
|
625 |
73 |
4 |
1 |
not reported |
not reported |
|
1250 |
68 |
4 |
1 |
not reported |
not reported |
|
2500 |
38 |
4 |
1 |
not reported |
not reported |
|
3600 |
31 |
1 |
0 |
not reported |
not reported |
|
ENU (50) |
53 |
35 |
9 |
not reported |
not reported |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 4: Second Experiment - 3 h exposure - With Metabolic Activation |
|
|
||||
Concentration (µg/mL) |
Cloning efficiency (%) |
Relative Total Growth (%) |
Mutants per 1E+4 surviving cells |
Mutation factor |
|
|
Day 0 |
Day 3 |
|
||||
0 (DMSO) |
100 |
100 |
100 |
2 |
1 |
|
156 |
83 |
95 |
62 |
1.95 |
0.98 |
|
313 |
84 |
101 |
70 |
1.84 |
0.92 |
|
625 |
82 |
105 |
65 |
1.79 |
0.9 |
|
1250 |
46 |
98.5 |
65 |
1.84 |
0.92 |
|
2500 |
38 |
88 |
37 |
1.93 |
0.97 |
|
3600 |
36 |
4 |
0 |
0 |
0 |
|
DMBA (3.3) |
28 |
52 |
27 |
37.3 |
18.65 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 5: Repeat fo Second Experiment - 24 h exposure - Without Metabolic Activation |
|
|||||
Concentration (µg/mL) |
Cloning efficiency (%) |
Relative Total Growth (%) |
Mutants per 1E+4 surviving cells |
Mutation factor |
|
|
Day 0 |
Day 3 |
|
||||
0 (DMSO) |
100 |
100 |
100 |
2.17 |
1 |
|
`2.5 |
102 |
100 |
62 |
2.16 |
1 |
|
5 |
100 |
114 |
151 |
2.02 |
0.93 |
|
10 |
92 |
95 |
145 |
1.97 |
0.91 |
|
20 |
105 |
100 |
60 |
2.11 |
0.97 |
|
40 |
82 |
95 |
103 |
2.17 |
1 |
|
80 |
69 |
111 |
97 |
1.82 |
0.84 |
|
160 |
25 |
44 |
16 |
2.32 |
1.07 |
|
320 |
11 |
31 |
6 |
1.94 |
0.89 |
|
ENU (50) |
16 |
31 |
9 |
51.8 |
23.87 |
|
ENU: N-ethyl-N-nitrosourea
DMBA: 7,12-dimethyl-1,2-benzanthracene
*: statistically significant with p < 0.01
#: could not be calculated as all wells contained mutant colonies
Table 1. Test results of the preliminary toxicity test
Treatment (µg/mL) |
Mitotic index (MI) |
|||||
Without S9 |
With S9 |
|||||
Individual values |
Relative mean MI (%) |
Individual values |
Relative mean MI (%) |
|||
0 |
4.8 |
5.3 |
100 |
3.8 |
2.9 |
100 |
313 |
4.8 |
4.3 |
90 |
3.1 |
3.5 |
99 |
625 |
4.3 |
4.1 |
83 |
2.9 |
3.5 |
96 |
1250 |
3.4 |
3.7 |
70 |
3.4 |
2.8 |
93 |
2500 |
3.7 |
3.3 |
69 |
2.1 |
1.8 |
58 |
5000 |
3.6 |
2.9 |
64 |
0.4 |
0.3 |
10 |
Mitotic index: percentage of cells at metaphase
Individual values: values for each of the duplicate
Relative mean MI: relative mean mitotic index for the duplicates
Table 2. Test results of the first main test (-S9: second repeat test, +S9: repeat test)
Treatment (µg/mL) |
S9 mix |
Relative mean MI (%) |
No. aberrant metaphases |
Number and types of aberrations |
Number of polyploid metaphases |
||
Gaps |
Breaks |
Exchanges |
|
||||
0 |
- |
100 |
1 / 0 |
2 / 1 |
1 / 0 |
|
1 / 0 |
1250 |
- |
110 |
1 / 1 |
0 / 2 |
2 / 1 |
|
|
2500 |
- |
75 |
1 / 4 |
1 / 3 |
1 / 4 |
|
|
5000 |
- |
70 |
2 / 3 |
2 / 1 |
2 / 4 |
|
|
Daunomycin (0.015 µg/mL) |
- |
91 |
17 / 14 ** |
9 / 5 |
20 / 14 |
2 / 1 |
|
0 |
2% |
100 |
0 / 0 |
1 / 2 |
0 / 0 |
|
|
625 |
2% |
67 |
0 / 1 |
0 / 0 |
0 / 1 |
|
|
1250 |
2% |
51 |
0 / 0 |
2 / 1 |
0 / 0 |
|
|
2500 |
2% |
42 |
0 / 1 |
1 / 1 |
0 / 1 |
|
|
Cyclophosphamide (6 µg/mL) |
2% |
20 |
33a /19b ** |
16 / 3 |
36 / 20 |
15 / 9 |
|
Relative mean: relative mean mitotic index for the duplicates
No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates
** Statistically significant with p < 0.01
a: only 59 scoreable metaphases
b: only 30 scoreable metaphases
The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)
Table 3. Test results of the second main test (repeat test)
Treatment (µg/mL) |
S9 mix |
Relative mean MI (%) |
No. aberrant metaphases |
Number and types of aberrations |
Number of polyploid metaphases |
||
Gaps |
Breaks |
Exchanges |
|
||||
0 |
- |
100 |
0 / 3 |
1 / 5 |
0 / 3 |
|
|
40 |
- |
84 |
1 / 4 |
3 / 6 |
1 / 4 |
|
1 / 0 |
80 |
- |
59 |
1 / 2 |
0 / 2 |
1 / 2 |
|
|
160 |
- |
49 |
0 / 2 |
3 / 1 |
0 / 2 |
|
0 / 1 |
Daunomycin (0.015 µg/mL) |
- |
102 |
13 / 14 ** |
6 / 9 |
13 / 13 |
1 / 1 |
|
0 |
4% |
100 |
2 / 1 |
3 / 2 |
1 / 1 |
1 / 0 |
|
625 |
4% |
66 |
2 / 4 |
3 / 1 |
5 / 1 |
|
0 / 1 |
1250 |
4% |
61 |
1 / 1 |
3 / 4 |
1 / 1 |
|
|
2500 |
4% |
33 |
5 / 1 |
3 / 0 |
3 / 4 |
|
|
Cyclophosphamide (6 µg/mL) |
4% |
77 |
36 / 33 ** |
8 / 8 |
37 / 33 |
11 / 11 |
|
Relative mean: relative mean mitotic index for the duplicates
No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates
** Statistically significant with p < 0.01
a: only 59 scoreable metaphases
b: only 30 scoreable metaphases
The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)
Table 4. Historical Data (n = 9 previous study)
Treatment (µg/mL) |
S9 mix |
Frequency of metaphases with aberrant chromosomes excluding gaps (%) |
Number of cultures |
|||
Mean |
SD |
Mimimum |
Maximum |
|
||
Negative control |
- |
0.8 |
0.8 |
0 |
3 |
32 |
Daunomycin (0.015 µg/mL) |
- |
15.1 |
7.4 |
7 |
34 |
32 |
Negative control |
+ |
0.7 |
0.9 |
0 |
3 |
32 |
Cyclophosphamide (6 µg/mL) |
+ |
43.0 |
13.6 |
23 |
70 |
32 |
Table 1. Effects of the test substance on viability and chromosome aberrations.
Test item |
Concentration |
Cell viability |
Aberrant cells in % |
|
|
in µg/mL |
in % |
Numerical |
Structural including gaps |
Exposure period 6h without S9 mix |
||||
Untreated |
-- |
-- |
0.0 |
0.0 |
Solvent |
-- |
100 |
0.0 |
1.5 |
MMC |
0.010 |
105.5 |
0.0 |
39.5 |
Test substance |
0.020 |
101.0 |
0.5 |
0.0 |
0.039 |
98.0 |
1.0 |
1.5 |
|
0.078 |
88.0 |
0.0 |
0.0 |
|
0.156 |
82.5 |
0.5 |
1.0 |
|
Exposure period 6h with S9 mix |
||||
Untreated |
-- |
-- |
0.0 |
2.0 |
Solvent |
-- |
100 |
0.0 |
0.5 |
B(a)P |
7.000 |
96.5 |
0.0 |
28.5 |
Test substance |
0.020 |
96.0 |
0.0 |
2.0 |
0.039 |
92.0 |
0.0 |
0.0 |
|
0.078 |
86.0 |
1.0 |
0.5 |
|
0.156 |
84.5 |
0.5 |
1.0 |
|
Exposure period 24h without S9 mix |
||||
Untreated |
-- |
-- |
1.0 |
0.5 |
Solvent |
-- |
100 |
0.5 |
0.0 |
MMC |
0.050 |
102.0 |
0.0 |
41.5 |
Test substance |
0.078 |
90.5 |
0.0 |
0.5 |
0.156 |
82.5 |
0.0 |
1.0 |
|
0.313 |
76.0 |
1.0 |
1.0 |
|
0.625 |
66.0 |
0.0 |
0.5 |
|
1.250 |
66.0 |
0.5 |
2.0 |
|
2.500 |
84.5 |
0.5 |
0.0 |
|
5.000 |
92.0 |
0.0 |
1.0 |
|
Exposure period 48h without S9 mix |
||||
Untreated |
-- |
-- |
0.0 |
0.0 |
Solvent |
-- |
100 |
0.5 |
0.0 |
MMC |
0.050 |
94.0 |
0.0 |
58.5 |
Test substance |
0.078 |
99.0 |
0.0 |
1.0 |
0.156 |
86.5 |
0.0 |
0.5 |
|
0.313 |
74.5 |
1.0 |
0.0 |
|
0.625 |
70.0 |
0.0 |
0.0 |
|
1.250 |
79.5 |
0.5 |
0.0 |
|
2.500 |
87.5 |
0.5 |
0.0 |
|
5.000 |
92.5 |
0.5 |
1.0 |
|
MMC: Mitomycin
B(a)P: Benz(a)pyrene
Table 1:Summary of results of the chromosome aberration study with Castor oil*
|
S9 |
Harvest time (h) |
Conc. in μg/mL |
Cells scored |
No of aberrations |
Aberrations /Cell |
Percent Cells with aberrations |
DMSO |
- |
12 |
|
200 |
2 |
0.01 |
1.0 |
Test item |
- |
12 |
1600 |
200 |
1 |
0.01 |
0.5 |
Test item |
- |
12 |
3000 |
200 |
2 |
0.01 |
1.0 |
Test item |
- |
12 |
5000 |
200 |
1 |
0.01 |
0.5 |
Pos. control (MMC) |
- |
12 |
0.0625 |
200 |
48 |
0.24 |
15.5 |
DMSO |
+ |
13 |
|
200 |
3 |
0.02 |
1.5 |
Test item |
+ |
13 |
1600 |
200 |
4 |
0.02 |
2.0 |
Test item |
+ |
13 |
3000 |
200 |
4 |
0.02 |
2.0 |
Test item |
+ |
13 |
5000 |
200 |
3 |
0.02 |
1.5 |
Pos. control (CP) |
+ |
13 |
2.5 |
200 |
44 |
0.22 |
16.0 |
MMC = Mitomycin C
CP = Cyclophosphamide
*In the absence of S9, cells were incubated with study compound or solvent for 10 h at 37°C. Cells were then washed and fresh medium containing colcemid was added for an additional 3 h followed by harvest. In the presence of S9, cells were incubated with study compound or solvent for 2 h at 37°C. Cells were then washed, medium without test compound was added, and incubation was continued for 10 h. Colcemid was added for the last 3 h of incubation before harvest.
Table 1:Summary of results of the chromosome aberration study in CHO cells treated with SALATRIM 23CA Lot A014
|
S9 |
Conc. in ¿g/mL |
Cells scored |
mitotic index (%) |
cells with abnormal chromosomes (%) |
cells with chromatid deletions /exchanges (%) |
structural aberrations per cell |
corn oil** |
- |
1000 |
100 |
9.6 |
2 |
2/0 |
0.02 |
MMS |
- |
20 |
100 |
4.7 |
18* |
13/5 |
0.22 |
Acetone |
- |
0 |
100 |
5.9 |
4 |
3/0 |
0.04 |
Test item |
- |
250 |
100 |
4.8 |
2 |
1/0 |
0.01 |
Test item |
- |
500 |
100 |
5.0 |
4 |
3/0 |
0.06 |
Test item |
- |
1000 |
100 |
4.4 |
4 |
3/0 |
0.05 |
corn oil** |
+ |
1000 |
100 |
4.7 |
5 |
4/0 |
0.05 |
CP |
+ |
50 |
100 |
0.4 |
26* |
16.2/6.1 |
0.42 |
Acetone |
+ |
0 |
100 |
3.6 |
4 |
3/0 |
0.06 |
Test item |
+ |
250 |
100 |
3.9 |
0 |
0/0 |
0.00 |
Test item |
+ |
500 |
100 |
3.9 |
2 |
2/0 |
0.03 |
Test item |
+ |
1000 |
100 |
5.1 |
3 |
3/0 |
0.03 |
*p<0.05
**corn oil was tested in an independent experiment
Table 3: Results of the HPRT Mammalian Cell Gene Mutation Assay of SALATRIM 23CA lot A014
with and without Metabolic Activation*
SALATRIM Dose (¿g/mL) |
without metabolic activation |
with metabolic activation |
||
rel. cloning efficiency (% of control) |
mutant frequency (per 106cells) |
rel. cloning efficiency (% of control) |
mutant frequency (per 106cells) |
|
0 |
100 |
15 |
100 |
10 |
31.3 |
96 |
7 |
94 |
7 |
62.5 |
100 |
18 |
84 |
6 |
125 |
89 |
11 |
93 |
10 |
250 |
90 |
17 |
97 |
15 |
500 |
78 |
12 |
69 |
9 |
1000 |
86 |
17 |
75 |
10 |
corn oil 1000** |
91 |
6 |
79 |
15 |
EMS200 |
56 |
170 |
- |
- |
3MC 5 |
- |
|
79 |
170 |
*Data represent the mean of two cultures with the exception of the 0 dose (solvent control), which represents the mean of triplicate cultures.
**Data from a independent experiment with corn oil as test substance instead of SALATRIM
EMS= Ethyl methanesulfonate; positive control for the assay without metabolic activation.
3MC = 3-Methylcholanthrene; positive control for the assay with metabolic activation.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro genotoxicity:
Several Fatty Acid Glycerides Category members were tested for their genotoxic potential in bacteria (Ames Test). In all testings Fatty Acid Glycerides were shown to be non-mutagenic in bacteria.
Salmonella typhimurium strains TA 97, TA 98 and TA100 were treated with the test material Glycerides, C12-18 (CAS No. 67701-26-2) diluted in EGDME using the Ames plate incorporation method according to OECD Guideline No. 471 (Kennelly, 1987). Test substance concentrations of 0, 1.6, 8, 40, 200, 1000 µg/plate were tested in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9). Limited solubility and precipitation of the test substance was observed at 1000 µg/plate, but at this dose the precipitated test agent did not interfere with the counting of colonies. Thus, 1000 µg/plate was chosen as the highest concentration fur mutation testing. The test material caused no cytotoxicity up to the highest, precipitating dose. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with the test material Glycerides, mixed decanoyl and octanoyl (CAS No. 73398-61-5) diluted in acetone according to EU Method B.13/14 (Schöberl, 1994). Test substance concentrations of 0, 8, 40, 200, 1000, 5000 µg/plate were tested in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9). Precipitation of the test substance was observed at 5000 µg/plate. The test material caused no cytotoxicity up to the highest, precipitating dose. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with the test material Castor oil, hydrogenated (CAS No. 8001-78-3) diluted in Tween 80/ bi-distilled water using the plate incorporation method according to OECD Guideline 471 (Banduhn, 1990). Test substance concentrations of 0, 8, 40, 200, 1000, 5000 µg/plate and 0, 6.25, 25, 100, 400, 1600 µg/plate (repetition test) were tested in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9). Precipitation of the test substance was observed, starting at a concentration of 200µg/plate. The test material caused no cytotoxicity up to the highest dose. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with the test material Glycerides, C14-18 and C16-22-unsatd. mono- and di- (CAS No. 91744-13-7) diluted in Tween 80/ bi-distilled water using the plate incorporation method according to OECD Guideline 471 (Banduhn, 1990). Test substance concentrations of 0, 8, 40, 200, 1000, 5000 µg/plate were tested in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9). The test material caused cytotoxicity in Salmonella strain TA 1535 at 5000 ¿g/plate. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
An in vitro mammalian chromosome aberration test was performed with Castor oil (CAS No. 8001-79-4) in Chinese hamster Ovary (CHO) cells (Irwin, NTP report 1992). The occurrence of chromosome aberrations was investigated in the presence and absence of metabolic activation (rat liver S9-mix) with test substance concentrations of 0, 1600, 3000, 5000 µg/mL diluted with DMSO. Castor oil did not induce a significant increase in the number of phases with aberrations at any preparation time and dose level. No relevant cytotoxic effects were reported. Positive controls significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay. In conclusion, Castor oil did not induce chromosome aberrations in Chinese hamster Ovary cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.
An in vitro Sister Chromatid Exchange Assay was performed with Castor oil (CAS No. 8001-79-4) in Chinese hamster Ovary (CHO) cells (Irwin, NTP report 1992). The occurrence of sister chromatid exchanges was investigated in the presence and absence of metabolic activation (rat liver S9-mix) with test substance concentrations of 0, 160, 500, 1600 and 5000 µg/mL diluted with DMSO. Castor oil did not induce a significant increase in the number of sister chromatid exchanges at any dose level. No relevant cytotoxic effects were reported. Positive controls significantly increased the rate of sister chromatid exchanges indicating the sensitivity of the assay.
A chromosome aberration study was performed with glycerides, C8 -18 and C18 -unsatd. mono- and di, acetates (CAS No. 91052 -13 -0) according to OECD guideline 473 with CHU/IU cells (Seki, 2010). The occurrence of chromosome aberrations was investigated in the presence and absence of metabolic activation (rat liver S9-mix) with test substance concentrations of 0.02, 0.39, 0.078 and 0.156 mg/mL for 6 hours with and without S9-mix and 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5.0 mg/mL for 24 and 48 hours without S9-mix diluted in acetone. The test substance did not induce a significant increase in the number of aberrations at any preparation time and dose level. No relevant cytotoxic effects were reported. The test substance precipitated at concentration ¿ 0.078 µg/mL. Positive controls significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay. In conclusion, the test substance did not induce chromosome aberrations in CHU/IU cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.
Structural analogue read-across from Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS No. 91052-13-0) for mammalian toxicity was judged to be justified for the following reasons: This read across substance is also a glyceride, containing mainly C12-14 fatty acid and actetate moiety as well as glycerol. It¿s an organic liquid with a pour point of -8 °C and a melting point of 357.85 °C and a vapour pressure < 0.01 Pa at room temperature. In contrast to the glycerides of the fatty acid glyceride category, it has a higher water solubility of 8.75 mg/L, which might influence its environmental distribution, but not the mammalian metabolism upon systemic uptake. Therefore it is expected to feed into the same mammalian physiological pathways as the members of the fatty acid glyceride category, like citric acid cycle, sugar synthesis and lipid synthesis. These processes are described in detail within the category justification.
An in vitro mammalian chromosome aberration test was performed with the SALATRIM (short- and long-chain acyl triglyceride molecules) family of triacylglycerols in Chinese hamster Ovary (CHO) cells (Hayes, 1994). The occurrence of chromosome aberrations was investigated in the presence and absence of metabolic activation (rat liver S9-mix) with test substance concentrations of 0, 250, 500 and 1000 µg/mL diluted with acetone. The high dose was limited by the low solubility of the fats in the assay medium. The test substance did not induce a significant increase in the number of phases with aberrations at any preparation time and dose level. No relevant cytotoxic effects were reported. Positive controls significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay. In conclusion, the SALATRIM family of triacylglycerols did not induce chromosome aberrations in Chinese hamster Ovary cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.
An in vitro mammalian cell gene mutation assay was performed with the SALATRIM (short- and long-chain acyl triglyceride molecules) family of triacylglycerols in Chinese hamster Ovary (CHO) cells (Hayes, 1994). Gene mutations in the HPRT locus were investigated in the presence and absence of metabolic activation (rat liver S9-mix) with test substance concentrations of 0, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL diluted with acetone. The high dose was limited by the low solubility of the fats in the assay medium. The test substance did not induce a significant increase in the mutant frequency at any preparation time and dose level. No relevant cytotoxic effects were reported. Positive controls significantly increased mutant frequency indicating the sensitivity of the assay. In conclusion, the SALATRIM family of triacylglycerols did not induce the mutant frequency in Chinese hamster Ovary cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.
In vivo genotoxicity:
An in vivo Mammalian Erythrocyte Micronucleus Test was performed with the SALATRIM (short- and long-chain acyl triglyceride molecules) family of triacylglycerols in Crl:CD BR VAF/Plus rats according to OECD Guideline 474 (Hayes, 1994). Groups of 20 male and 20 female animals were exposed to either of two SALATRIM fats or corn oil at 10% (w/w) of the diet (equivalent to 7000 mg/kg bw/day) for at least 13 weeks. Erythrocytes were scored to determine the frequency of micro-nucleated erythrocytes. No signs of systemic toxicity in any of the treated animals were observed. No increases in the frequency of micronuclei in polychromatic erythrocytes of the femoral bone marrow of Crl:CD BR VAF/Plus rats exposed to 7000 mg/kg bw SALATRIM occurred. Because these data were collected from a 13-week sub-chronic toxicity study, a positive control for micronuclei formation was not included.
An in vivo Mammalian Erythrocyte Micronucleus Test was performed with Castor oil (CAS No. 8001-79-4) in B6C3F1 mice similar to OECD Guideline 474 (Irwin, NTP report 1992). 10 animals per group were treated with test substance concentrations of 0, 0.62, 1.25, 2.50, 5.00, 10.0 % (w/w) in the diet by oral feeding for 13 weeks (approx. 0, 917, 2022, 3800, 7823, 15017 mg/kg bw/day). Blood smears were prepared from peripheral blood samples obtained by cardiac puncture of dosed and control animals at the termination of the 13 week study. At least 2000 PCE and 10000 NCE from each animal were scored to determine the frequency of micro-nucleated erythrocytes. No signs of systemic toxicity in any of the treated animals were observed. No increases in the frequency of micronuclei in Peripheral Blood Erythrocytes of B6C3F1 mice exposed to Castor Oil in doses up to approx. 15000 mg/kg bw fed for 13 weeks occurred, whereas the positive control substance (0.2 % urethane) significantly increased the number of normochromatic and polychromatic erythrocytes with micronuclei in three control animals.
An in vivo Mammalian Erythrocyte Micronucleus Test was performed with Glycerides, C16-18 and C18-hydroxy mono- and di- (CAS No. 91845-19-1) in CFW 1 mice according to OECD Guideline 474 (Wallat, 1985). 7 animals received a single dose of the test substance diluted in peanut oil at a concentrations of 10000 mg/kg bw by oral gavage. 24 hours after application the femoral bone marrow was taken from both femurs. 1000 erythrocytes per animal were scored to determine the frequency of micro-nucleated erythrocytes. No signs of systemic toxicity in any of the treated animals were observed. No increases in the frequency of micronuclei in polychromatic erythrocytes of the femoral bone marrow of CFW 1 mice exposed to 10000 mg/kg bw Glycerides, C16-18 and C18-hydroxy mono- and di- occurred, whereas the positive control substance (10 mg cyclophosphamide/kg bw) significantly increased the number of polychromatic erythrocytes with micronuclei.
Considering the clearly negative results of all available in vitro and in vivo genotoxicity testings of Fatty Acid Glycerides and the proven physiological function of triglycerides, it can be assumed that all members of this category are not genotoxic, neither in vitro nor in vivo.
Justification for classification or non-classification
According to DSD (67/548/EEC) or CLP (1272/2008/EC) classification criteria for genetic toxicity, no classification is required.
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