Registration Dossier
Registration Dossier
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EC number: 306-523-3 | CAS number: 97281-24-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Genetic toxicity in vitro:
Bacterial mutagenicity (OECD 471, Ames): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538 and in E. coli WP2 uvrA (data from registered substance and RA from CAS 85186-86-3)
Mammalian cytogenicity (OECD 473, CA, WoE): negative in cultured peripheral human lymphocytes and Chinese hamster ovary cells (CHO) with and without metabolic activation (RA from CAS 68855-18-5 and CAS 11138-60-6)
Mammalian mutagenicity (OECD 476, MLA): negative in mouse lymphoma L5178Y cells with and without metabolic activation (RA from CAS 68855-18-5)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 01 Mar - 11 Mar 1988
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP - Guideline study with acceptable restrictions (no analytical purty reported; no E.coli strain tested)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in 1983
- Deviations:
- yes
- Remarks:
- no analytical purty reported; no E.coli strain tested
- 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:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Experiment I+II:
- 8, 40, 200, 1000 and 5000 µg/plate (with and without metabolic activation) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Tween 80 / water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: With and Without S9: sodium azide (2 µg/plate) for TA100 and TA1535; 9-aminoacridine (80 µg/plate) for TA1537; 4-nitro-o-phenylendiamine (40 µg/plate) for TA98 and TA1538. With and without S9: 2 aminoanthracene (2-AA; 2.5 or 5 µg/plate) for all strains
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: triplicates each in two independent experiments - Evaluation criteria:
- The test material was considered positive in this test system if the following criteria were met:
- The plate background from the non revertant bacteria showed no growth reduction in comparison to the correspondent negative control.
- The spontaneous mutation rate of the strains tested was within the characteristic range of the spontaneous mutation.
- The positive controls showed mutation rates that exceeded the control values of the TA100 at least by a factor 2.0 and the other tester strains, at least by a factor 3.0.
- For the test item, two-fold increases in mean revertant numbers were observed at more then one dose level in TA 100. For the other strains, an increase in the mutation rate greater than three-fold in comparison to the correspondent negative control was observed. - Statistics:
- Mean values and standard deviation were calculated.
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- 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
- Positive controls validity:
- valid
- Conclusions:
- negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 Jul - 19 Dec 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP - Guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted in 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- The department of health of the government of the United Kingdom
- 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 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL) and 10% donor horse serum (giving R10 media. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) were used during the course of the study
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbonel/beta-naphthoflavone
- Test concentrations with justification for top dose:
- Experiment I:
- 1.6, 3.2, 6.41, 12.81, 25.63, 51.25, 76.88, and 102.5 µg/mL (with and without metabolic activation)
Experiment II:
- 1.6, 3.2, 6.41, 12.81, 25.63, 51.25, 76.88, and 102.5 µg/mL (without metabolic activation)
- 3.2, 6.41, 12.81, 25.63, 51.25, 76.88, 102.5, and 205.0 µg/mL (with metabolic activation) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Remarks:
- cyclophosphamide, 2 µg/mL, +S9; ethylmethanesulphonate 400 µg/mL in Exp1 and 150 µg/mL in Exp 2, –S9.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
1st experiment: 4 h exposure with and without S9 mix.
2nd experiment: 4 h exposure with S9 mix and 24 h with and without S9 mix.
- Expression time (cells in growth medium): 2 days after the end of the reatment, cells were plated for determination of the mutation frequency in 96-well microtitre plates containing TFT selective medium. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium. The microtitre plates were incubated for 10 to 14 days.
- Selection time (if incubation with a selection agent): 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12-14 days
SELECTION AGENT (mutation assays): 4µg/mL trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: duplicates each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- See any other information on materials and methods incl. tables
- Statistics:
- The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson, W.D. et al., 1989).
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment I: ≥51.25 µg/mL (4 h, -S9-mix); Experiment II: ≥25.63 µg/mL (24 h, -S9-mix)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: there was no marked change in pH when the test item was dosed into media.
- Effects of osmolality: the osmolality did not increase by more than 50 mOsm in the solubility test.
RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed on cell cultures at 5 x 10E5 cell/mL, using a 4h exposure-period with both and without S9, and at 1.5 x 10E5 cells/mL using a 24h exposure-period without S9. The dose range used in the preliminary toxicity test was 6.41 to 1640 µg/mL for all three of the exposure groups. Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
1) Maximum recommended dose level, 5000 µg/mL or 10 mM.
2) The presence of excessive precipitate where no test item-induced toxicity was observed.
3) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required).
COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for L5178Y cell line at the TK +/- locus.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) values that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.
In the Experiment 1 there was evidence of marked dose-related toxicity following exposure to the test item in the absence of metabolic activation, as indicated by the RTG and % RSG values. In the presence of metabolic activation there was no evidence of the test item induced toxicity which varied from the toxicity observed in the preliminary toxicity test. There was no evidence of a marked reduction in viability (%V) in the absence or presence of metabolic activation therefore indicating that residual toxicity had not occurred. Near to optimum levels of toxicity were achieved in the absence of metabolic activation only. The test item proved to be non-toxic in the presence of metabolic activation, therefore optimum toxicity could not be achieved. Acceptable levels of toxicity were seen with both positive control substances. In Experiment 2 there was evidence of marked toxicity following exposure to the test item in the absence of metabolic activation, as indicated by RTG and %RSG values. In the presence of metabolic activation there was evidence of slight test item induced toxicity at the top dose level (205 µg/mL). Based on the toxicity results from the preliminary toxicity testing and Experiment 1 it was considered that the test item had been tested up to the limit of its exposure to cells and therefore, adequately tested. There was evidence of slight reductions in viability (%V) in the absence of metabolic activation, therefore indicating that residual toxicity had occurred. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the absence of metabolic activation only. The excessive toxicity observed at and above 76.9 µg/mL in the absence of metabolic activation resulted in these dose levels not being plated for viability of 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.
OTHER:
Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations. - Conclusions:
- negative
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 03 Nov - 05 Dec 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP - Guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- (adopted 2008)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Harlan Laboratories Ltd., ShaThe department of health of the government of the United Kingdom
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (S. typhimurium strains); trp operon (E. coli strain)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (10 % liver S9 mix in standard co-factors), prepared from the livers of rats treated with Phenobarbitone/ß-Naphthoflavone
- Test concentrations with justification for top dose:
- Experiment I+II:
- 50, 150, 500, 1500, and 5000 µg/plate (with and without metabolic activation) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test substance was fully miscible in acetone at 100 mg/mL in solubility pre-tests. - Untreated negative controls:
- yes
- Remarks:
- concurrent untreated control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Without S9: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG, 2, 3 or 5 µg/plate for WP2uvrA, TA100, or TA1535), 9-Aminoacridine (9AA, 80 µg/plate for TA1537), 4-Nitroquinoline-1-oxide (4NQO, 0.2 µg/plate for TA98)
- Untreated negative controls:
- yes
- Remarks:
- concurrent untreated control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: with S9: 2-Aminoanthracene (2AA, 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA), Benz(a)pyrene (BP, 5 µg/plate for TA98)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: three replicates/concentration or control
DETERMINATION OF FREQUENCY OF REVERTANT COLONIES: Domino colony counter
DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn - Evaluation criteria:
- A result was considered as positive, when the following criteria were met:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response) - Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: 1. no cytotoxicity (plate incorparation assay); 2. cytotoxicity at 5000 µg/plate in TA100, TA1535 and TA1537 without S9 (pre-incubation assay)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
A preliminary test was carried out to determine the toxicity of the test item using TA100 and WP2uvrA. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. In addition, a vehicle control (acetone) and a sterility control were run in parallel.
The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). - Conclusions:
- negative
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- Please refer to the Additional Information field in the endpoint study summary
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and 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
- Positive controls validity:
- valid
- Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- no cytotoxicity (plate incorparation assay); 2. cytotoxicity at 5000 μg/plate in TA100, TA1535 and TA1537 without S9 (pre-incubation assay)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: BASF, 1988d
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- Please refer to the Additional Information field in the endpoint study summary
- 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:
- cytotoxicity
- Remarks:
- Experiment I: ≥51.25 µg/mL (4 h, -S9-mix); Experiment II: ≥25.63 µg/mL (24 h, -S9-mix)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: Croda, 2013
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 22 May - 28 Oct 1996
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP - Guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adpoted in 1995
- Deviations:
- yes
- Remarks:
- Only basic data on test substance given
- 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):
- - Type and identity of media: McCoy´s 5A medium supplemented with 10% FBS, 100 units penicillin and 100 µg streptomycin/ml and 2 mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9-mix (Spraque-Dawley)
- Test concentrations with justification for top dose:
- Preliminary cytotoxicity test:
- 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/mL (with and without metabolic activation)
Chromosomal abberation assay:
- 157, 313, 625, 1250, 2500, 5000 µg/mL (with and without metabolic activation)
Concentrations 157 and 313 µg/mL were not evaluated for CA - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: mitomycin C, 0.08 and 0.15 µg/mL, -S9; cyclophosphamide 10 µg/mL, +S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 and 20 h without S9 activation and 4 h with activation.
STAIN (for cytogenetic assays): 5% Giemsa
NUMBER OF CELLS EVALUATED: A minimum of 200 metaphase spreads (100 per duplicate flask) at each concentration used. - Evaluation criteria:
- Criteria for cytotoxicity of the test substance: (1) cell growth inhibition relative to the solvent control
Criteria for chromosomal damage: (1) Number and types of aberrations found, the percentage of structurally and numerically damaged cells in the total population were counted. - Statistics:
- The frequency of structural aberrations per cell was calculated. The statistical analysis of the percent aberrant cells was performed with Fisher´s Exact Test. It was used to compare pair wise the percent aberrant cells of each treatment group with that of the solvent control. In the event of positive control, the Cochran-Armitage test was used to measure dose- responsiveness.
The dose response was estimated by linear regression curves. Difference in the sensitivity of mutagenic compounds was established by comparing the slopes of corresponding dose-response regression curves. For a comparison of mean values, Student's t test was used. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 625 and 5000 µg/mL (experiment I without metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 8 in the highest dose
- Effects of osmolality: 285 in the highest dose
RANGE-FINDING/SCREENING STUDIES: Yes, cell growth inhibition of 83% without and 3% with metabolic activation at the highest dose tested (5000 µg/mL).
COMPARISON WITH HISTORICAL CONTROL DATA: Yes - Conclusions:
- negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 16 Aug 2012- 24 Jan 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP - Guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted in 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted in 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- The department of health of the government of the United Kingdom
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Eagle´s minimal essential medium with HEPES buffer (MEM) supplemented with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: 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 phenobarbonel/beta-naphthoflavone
- Test concentrations with justification for top dose:
- Experiment I+II:
- 12.5, 25, 50, 100, 200 and 400 µg/mL (with and without metabolic activation) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone (test substance), dimethyl sulphoxide (cyclophosphamide), and Minimal Essential Medium (mytomicin C)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Remarks:
- +S9, cyclophosphamide, 5 µg/mL in dimethyl sulphoxide; -S9; mitomycin C, 0.4 µg/mL in Exp1, and 0.2 µg/mL in Exp 2, –S9 in Minimal Essential Medium.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4h treatment: 24 h; 24 h treatment: 24 h
SPINDLE INHIBITOR (cytogenetic assays): democolcine (Colcemid 0.1 µg/mL)
STAIN (for cytogenetic assays): Giemsa 5%
NUMBER OF REPLICATIONS: duplicate in two independent experiments
NUMBER OF CELLS EVALUATED: 100 well-spread metaphases per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 lymphocyte cell nuclei
OTHER EXAMINATIONS:
- Determination of polyploidy: yes - Evaluation criteria:
- A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increased in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
- Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, when necessary, with the concurrent vehicle control value using the Fisher´s Exact test.
- Key result
- Species / strain:
- lymphocytes: human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In Exp , -S9, there was a plateau in toxicity between 50 and 400 µg/mL. In Exp 2 at 400 µg/mL (inhibition 45% of mitotic index).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: there was no significant change in pH when the test item was dosed into media
- Effects of osmolality: the osmolality did not increase by more than 50 mOsm
RANGE-FINDING/SCREENING STUDIES: Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test. - Conclusions:
- negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Please refer to the Additional Information field in the endpoint study summary
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- lymphocytes: human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In Exp , -S9, there was a plateau in toxicity between 50 and 400 μg/mL. In Exp 2 at 400 μg/mL ( inhibition 45% of mitotic index).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 625 and 5000 μg/mL (experiment I without metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Croda, 2013a
Referenceopen allclose all
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 |
TA1538 |
TA98 |
TA1537 |
|||||||||||
– |
0 |
70.3 ± 8.7 |
8.6 ± 1.8 |
15.3 ± 2.2 |
15.8 ± 5.8 |
7.8 ± 1.1 |
|||||||||
– |
8 |
70.3 ± 9.2 |
6.0 ± 1.7 |
6.0 ± 1.0 |
15.3 ± 3.7 |
3.0 ± 1.0 |
|||||||||
– |
40 |
73.6 ± 6.3 |
8.3 ± 2.0 |
5.3 ± 1.5 |
14.6 ± 1.5 |
5.3 ± 2.5 |
|||||||||
– |
200 |
77.0 ± 5.2 |
9.0 ± 1.0 |
6.0 ± 2.6 |
12.6 ± 3.2 |
5.0 ± 1.7 |
|||||||||
– |
1000 |
71.6 ± 2.0 |
8.3 ± 1.1 |
8.6 ± 1.5 |
13.3 ± 1.5 |
7.0 ± 2.6 |
|||||||||
– |
5000 |
78.6 ± 13.0 |
5.0 ± 1.7 |
7.6 ± 2.8 |
11.0 ± 4.0 |
3.3 ± 1.1 |
|||||||||
Positive controls, –S9 |
Name |
NaA |
2AA |
NaA |
2AA |
4NOPD
|
2AA |
4NOPD
|
2AA |
9AA
|
2AA |
||||
Concentrations (μg/plate) |
2.0 |
5.0 |
2.0 |
2.5 |
40.0 |
5.0 |
40.0 |
5.0 |
80 |
2.5 |
|||||
Mean No. of colonies/plate (average of 3) |
235 ± 30.6 |
69.6 ± 1.5 |
255.3 ± 24.8 |
6.3 ± 0.57 |
844.0 ± 54.6 |
9.6 ± 4.7 |
370.0 ± 14.7 |
16.6 ± 3.5 |
146.0 ± 9.6 |
4.6 ± 1.5 |
|||||
+ |
0 |
83.5 ± 11.5 |
10.5 ± 2.5 |
14.5 ± 2.0 |
20.8 ± 3.9 |
7.6 ± 0.51 |
|||||||||
+ |
8 |
76.3 ± 2.0 |
9.6 ± 1.1 |
14.0 ± 3.0 |
17.6 ± 3.5 |
5.3 ± 2.5 |
|||||||||
+ |
40 |
74.6 ± 5.6 |
8.3 ± 1.1 |
13.6 ± 1.5 |
15.3 ± 1.5 |
6.3 ± 2.5 |
|||||||||
+ |
200 |
82.6 ± 0.57 |
10.0 ± 1.0 |
12.6 ± 2.8 |
19.6 ± 0.6 |
5.6 ± 3.0 |
|||||||||
+ |
1000 |
90.3 ± 5.5 |
13.6 ± 4.7 |
11.3 ± 3.2 |
19.6 ± 6.1 |
7.3 ± 3.2 |
|||||||||
+ |
5000 |
80.3 ± 16.1 |
12.6 ± 1.5 |
9.0 ± 1.7 |
22.3 ± 3.7 |
7.6 ± 0.6 |
|||||||||
Positive controls, + S9 |
Name |
NaA |
2AA |
NaA |
2AA |
4NOPD
|
2AA |
4NOPD
|
2AA |
9AA
|
2AA |
||||
Concentrations (μg/plate) |
2.0 |
5.0 |
2.0 |
2.5 |
40.0 |
5.0 |
40.0 |
5.0 |
80 |
2.5 |
|||||
Mean No. of colonies/plate (average of 3) |
125.3 ± 9.6 |
1292.6 ± 100.4 |
44.0 ± 8.8 |
223.6 ± 47.0 |
793.6 ± 50.0 |
441.0 ± 49.3 |
450.0 ± 32.0 |
643.3 ± 137.3 |
61.6 ± 2.8 |
75.6 ± 7.5 |
|||||
Table 2: Test Results of Experiment 2 (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 |
TA1538 |
TA98 |
TA1537 |
|||||||||||
– |
0 |
90.0 ± 6.8 |
9.5 ± 4.7 |
11.1 ± 3.8 |
16.8 ± 1.9 |
5.8 ± 1.1 |
|||||||||
– |
8 |
86.0 ± 6.2 |
9.0 ± 2.0 |
7.6 ± 3.0 |
17.6 ± 0.6 |
5.3 ± 3.2 |
|||||||||
– |
40 |
89.0 ± 14.7 |
11.6 ± 2.0 |
7.0 ± 3.4 |
15.6 ± 4.0 |
6.6 ± 2.0 |
|||||||||
– |
200 |
100.6 ± 14.2 |
5.0 ± 2.0 |
9.6 ± 2.5 |
12.0 ± 2.6 |
4.3 ± 1.1 |
|||||||||
– |
1000 |
90.0 ± 10.8 |
10.0 ± 2.6 |
12.0 ± 5.1 |
14.0 ± 3.4 |
5.6 ± 3.0 |
|||||||||
– |
5000 |
97.0 ± 17.4 |
7.3 ± 2.0 |
8.0 ± 2.0 |
12.6 ± 3.0 |
5.6 ± 2.8 |
|||||||||
Positive controls, –S9 |
Name |
NaA |
2AA |
NaA |
2AA |
4NOPD
|
2AA |
4NOPD
|
2AA |
9AA
|
2AA |
||||
Concentrations (μg/plate) |
2.0 |
5.0 |
2.0 |
2.5 |
40.0 |
5.0 |
40.0 |
5.0 |
80 |
2.5 |
|||||
Mean No. of colonies/plate (average of 3) |
318.0 ± 22.2 |
104.0 ± 6.2 |
340.3 ± 14.5 |
10.3 ± 4.0 |
1009.0 ± 44.3 |
7.0 ± 1.7 |
441.6 ± 19.3 |
20.0 ± 1.7 |
211.6.± 20.5 |
7.3 ± 2.0 |
|||||
+ |
0 |
96.5± 15.8 |
10.3 ± 1.9 |
16.3 ± 3.1 |
17.1 ± 4.0 |
7.8 ± 2.4 |
|||||||||
+ |
8 |
94.3 ± 10.0 |
10.0 ± 2.6 |
17.0 ± 2.0 |
17.3 ± 1.1 |
7.3 ± 1.1 |
|||||||||
+ |
40 |
94.0 ± 4.3 |
10.6 ± 2.5 |
17.0 ± 4.5 |
16.6 ± 1.1 |
5.3 ± 0.6 |
|||||||||
+ |
200 |
105.0 ± 4.5 |
8.6 ± 3.0 |
13.6 ± 1.5 |
16.6 ± 4.0 |
7.0 ± 2.0 |
|||||||||
+ |
1000 |
108.6 ± 7.3 |
12.3 ± 5.0 |
14.3 ± 4.0 |
18.6 ± 2.5 |
6.3 ± 1.5 |
|||||||||
+ |
5000 |
112.0 ± 9.6 |
10.3 ± 1.1 |
9.60 ± 1.5 |
16.3± 7.0 |
4.6 ± 1.5 |
|||||||||
Positive controls, + S9 |
Name |
NaA |
2AA |
NaA |
2AA |
4NOPD
|
2AA |
4NOPD
|
2AA |
9AA
|
2AA |
||||
Concentrations (μg/plate) |
2.0 |
5.0 |
2.0 |
2.5 |
40.0 |
5.0 |
40.0 |
5.0 |
80 |
2.5 |
|||||
Mean No. of colonies/plate (average of 3) |
156.6 ± 14.2 |
884.6 ± 109.5 |
95.3 ± 6.4 |
114.3 ± 45.2 |
1204.6 ± 38.0 |
569.0 ± 62.0 |
635.3 ± 27.7 |
421.3 ± 30.0 |
88.3 ± 33.2 |
54.3 ± 16.0 |
|||||
9AA = 9-aminoacridine
2AA = 2-Aminoanthracene
NaA= Sodiumazide
4NOPD= 4-Nitro-o-phenylendiamin
Table 1: Experiment I - 4 h exposure - Without Metabolic Activation
Concentration |
Relative Total Growth [%] |
Relative Total Growth |
Mutation factor |
0 (acetone) |
100 |
1.00 |
181.34 |
1.6 |
96 |
-- |
-- |
3.2 |
97 |
-- |
-- |
6.41 |
91 |
1.03 |
207.35 |
12.81 |
88 |
0.98 |
175.16 |
25.63 |
74 |
0.83 |
189.77 |
51.25 |
43 |
0.42 |
220-94 |
76.88 |
35 |
0.40 |
194.97 |
102.5 |
28 |
0.47 |
139.22 |
EMS, 400 |
69 |
0.87 |
705.33 |
EMS: Ethyl methane sulphonate
Table 2: Experiment I - 4 h exposure - With Metabolic Activation
Concentration |
Relative Total Growth [%] |
Relative Total Growth |
Mutation factor |
0 (acetone) |
100 |
1.00 |
177.48 |
1.6 |
105 |
-- |
-- |
3.2 |
107 |
-- |
-- |
6.41 |
101 |
0.86 |
204.41 |
12.81 |
106 |
1.02 |
164.09 |
25.63 |
99 |
0.91 |
190.08 |
51.25 |
96 |
0.93 |
183.37 |
76.88 |
103 |
1.05 |
166.33 |
102.5 |
96 |
0.94 |
191.22 |
CP, 2 |
63 |
0.36 |
1320.55 |
CP: Cyclophosphamide
Table 3: Experiment II - 24 h Exposure - Without Metabolic Activation
Concentration |
Relative Total Growth [%] |
Relative Total Growth |
Mutation factor |
0 (acetone) |
100 |
1.00 |
173.24 |
1.6 |
91 |
0.89 |
159.79 |
3.2 |
87 |
0.83 |
165.80 |
6.41 |
80 |
0.89 |
142.67 |
12.81 |
76 |
0.89 |
127.24 |
25.63 |
49 |
0.63 |
158.76 |
51.25 |
15 |
0.27 |
162.70 |
76.88 |
3 |
|
|
102.5 |
2 |
|
|
EMS, 150 |
65 |
0.54 |
1563.99 |
EMS: Ethyl methane sulphonate
Table 4: Experiment II - 4 h Exposure – With Metabolic Activation
Concentration |
Relative Total Growth [%] |
Relative Total Growth |
Mutation factor |
0 (acetone) |
100 |
1.00 |
144.47 |
3.2 |
86 |
|
|
6.4 |
83 |
|
|
12.8 |
86 |
0.85 |
143.05 |
25.6 |
86 |
0.81 |
172.98 |
51.3 |
82 |
0.93 |
127.62 |
76.9 |
87 |
0.95 |
144.64 |
102.5 |
83 |
0.85 |
149.46 |
205 |
78 |
0.89 |
132.37 |
CP, 2 |
62 |
0.45 |
1059.59 |
CP: Cyclophosphamide
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 ± standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
– |
0 |
84 ± 2.1 |
14 ± 3.5 |
22 ± 2.3 |
30 ± 5.9 |
19 ± 4.6 |
– |
50 |
72 ± 14.2 |
17 ± 3.5 |
24 ± 2.6 |
30 ± 6.4 |
20 ± 5.0 |
– |
150 |
76 ± 2.3 |
13 ± 4.4 |
21 ± 8.1 |
31 ± 8.5 |
13 ± 3.5 |
– |
500 |
82 ± 6.9 |
18 ± 3.8 |
23 ± 8.7 |
23 ± 2.6 |
13 ± 3.5 |
– |
1500 |
82 ± 16.5 |
14 ± 5.9 |
15 ± 4.0 |
24 ± 8.3 |
10 ± 1.5 |
– |
5000 |
84 ± 2.6 |
12 ± 1.2 |
21 ± 7.5 |
25 ± 6.4 |
15 ± 2.1 |
Positive controls, –S9 |
Name |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
Concentrations (μg/plate) |
3 |
5 |
2 |
0.2 |
80 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
557 ± 12.1 |
96 ± 20.6 |
639 ± 25.1 |
107 ± 11.5 |
360 ± 52.9 |
|
+ |
0 |
88 ± 7.0 |
13 ± 3.1 |
33 ± 6.7 |
17 ± 2.5 |
14 ± 4.0 |
+ |
50 |
86 ± 3.6 |
12.± 2.5 |
29 ± 7.5 |
14 ± 1.0 |
14 ± 3.5 |
+ |
150 |
83 ± 7.0 |
13 ± 3.2 |
25 ± 10.1 |
19 ± 1.7 |
13 ± 3.2 |
+ |
500 |
97 ± 14.8 |
13 ± 3.5 |
24 ± 6.7 |
16 ± 3.5 |
7 ± 0.6 |
+ |
1500 |
97 ± 27.8 |
12 ± 2.3 |
28 ± 5.3 |
19 ± 7.6 |
17 ± 2.1 |
+ |
5000 |
87 ± 5.8 |
12 ± 2.0 |
29 ± 3.6 |
22 ± 2.5 |
13 ± 0.6 |
Positive controls, +S9 |
Name |
2AA |
2AA |
2AA |
BP |
2AA |
Concentrations (μg/plate) |
1 |
2 |
10 |
5 |
2 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
554 ± 36.4 |
412 ± 20.8 |
206 ± 11.5 |
114 ± 1.5 |
268 ± 19.5 |
|
ENNG = N-ethyl-N-nitro-N-nitrosoguanidine
4NQO = 4-nitroquinoline-N-oxide
9AA = 9-aminoacridine
2AA = 2-Aminoanthracene
BP = Benzo(a)pyrene
P = Precipitate
C = contaminated
Table 2. Test results of experiment 2 (pre-incubation)
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
– |
0 |
87 ± 2.1 |
16 ± 2.6 |
32 ± 4.6 |
20 ± 6.8 |
10 ± 1.7 |
– |
50 |
84 ± 14.3 |
17 ± 4.5 |
24 ± 3.0 |
23.± 4.4 |
12 ± 7.1 |
– |
150 |
78 ± 5.0 |
15 ± 2.6 |
22 ± 2.3 |
21 ± 4.5 |
10 ± 3.0 |
– |
500 |
95 ± 12.1 |
19 ± 5.3 |
33 ± 7.0 |
20 ± 2.3 |
11 ± 2.6 |
– |
1500 |
83 ± 5.0 |
17 ± 3.6 |
33 ± 4.5 |
22 ± 1.7 |
11 ± 2.6 |
– |
5000 |
83 ± 6.0 t |
9 ± 1.5 t |
34 ± 1.2 |
14 ± 4.0 |
12 ± 4.5 t |
Positive controls, –S9 |
Name |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
Concentrations (μg/plate) |
3 |
5 |
2 |
0.2 |
80 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
532 ± 3.2 |
170 ± 4.0 |
769 ± 54.8 |
127 ± 20.6 |
1319 ± 210.8 |
|
+ |
0 |
104 ± 14.5 |
21 ± 6.1 |
33 ± 3.2 |
12 ± 1.2 |
11 ± 1.2 |
+ |
50 |
99 ± 4.9 |
19 ± 12.2 |
28 ± 7.6 |
12 ± 1.7 |
9 ± 1.5 |
+ |
150 |
92 ± 1.2 |
20 ± 8.1 |
33 ± 3.1 |
13 ± 3.1 |
10 ± 2.1 |
+ |
500 |
109 ± 20.4 |
16 ± 2.6 |
29 ± 3.8 |
13 ± 1.7 |
10 ± 1.0 |
+ |
1500 |
94 ± 12.0 |
15 ± 2.5 |
36 ± 6.8 |
11 ± 0.6 |
10 ± 2.9 |
+ |
5000 |
100 ± 2.6 |
11 ± 0.0 |
34 ± 4.0 |
10 ± 3.1 |
7 ± 4.9 |
Positive controls, +S9 |
Name |
2AA |
2AA |
2AA |
BP |
2AA |
Concentrations (μg/plate) |
1 |
2 |
10 |
5 |
2 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1354 ± 153.5 |
357 ± 6.1 |
147 ± 5.3 |
286 ± 1.0 |
295 ± 1.7 |
|
ENNG = N-ethyl-N-nitro-N-nitrosoguanidine
4NQO = 4-nitroquinoline-N-oxide
9AA = 9-aminoacridine
2AA = 2-Aminoanthracene
BP = Benzo(a)pyrene
P = Precipitate
C = contaminated
t= citotoxic effects
Table 1: Chromosomal aberration – Summary
Treatment [µg/ml] |
S9 activation |
Treatment/ harvest time [h] |
Mitotic index |
Cells scored |
Aberrations/cell (mean±SD) |
Cells with aberrations [%] |
|
numerical |
structural |
||||||
Vehicle (Ethanol) |
- |
4/20 |
6.9 |
200 |
0.005±0.071 |
2.5 |
0.5 |
625 |
- |
4/20 |
5.9 |
200 |
0.000±0.000 |
2.0 |
0.0 |
1250 |
- |
4/20 |
5.9 |
200 |
0.025±0.186 |
4.0 |
2.0 |
2500 |
- |
4/20 |
6.5 |
200 |
0.025±0.186 |
2.5 |
2.0 |
5000 |
- |
4/20 |
4.4 |
200 |
0.000±0.000 |
0.5 |
0.0 |
MMC (0.08) |
- |
4/20 |
6.1 |
200 |
0.150±0.788 |
3.5 |
9.0* |
Vehicle (Ethanol) |
+ |
4/20 |
7.8 |
200 |
0.025±0.157 |
2.0 |
2.5 |
625 |
+ |
4/20 |
7.0 |
200 |
0.020±0.140 |
1.0 |
2.0 |
1250 |
+ |
4/20 |
6.9 |
200 |
0.035±0.184 |
3.0 |
3.5 |
2500 |
+ |
4/20 |
7.4 |
200 |
0.030±0.222 |
2.0 |
2.0 |
5000 |
+ |
4/20 |
7.7 |
200 |
0.010±0.100 |
2.5 |
1.0 |
CP (10) |
+ |
4/20 |
2.3 |
200 |
0.950±1.591 |
2.5 |
45.5* |
Vehicle (Ethanol) |
- |
20/20 |
7.6 |
200 |
0.020±0.140 |
1.0 |
2.0 |
625 |
- |
20/20 |
6.4 |
200 |
0.015±0.158 |
1.5 |
1.0 |
1250 |
- |
20/20 |
5.5 |
200 |
0.025±0.186 |
2.0 |
2.0 |
2500 |
- |
20/20 |
6.0 |
200 |
0.025±0.157 |
2.5 |
2.5 |
5000 |
- |
20/20 |
6.2 |
200 |
0.020±0.172 |
2.0 |
1.5 |
MMC (0.08) |
- |
20/20 |
5.8 |
200 |
0.220±0.513 |
1.0 |
18.0* |
MMC = Mitomycine C
CP = Cyclophosphamide
* = p≤0.01; Fisher´s exact test
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for read-across
There are only limited data available addressing the genetic toxicity of Fatty acids, C8-10 (even numbered), diesters with neopentyl glycol and di- and triesters with trimethylolpropane (CAS 97281-24-8). In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).
Having regard to the general rules for the read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a comparable pattern as a result of structural similarity, the substances heptanoic acid, ester with 2,2-dimethyl-1,3-propanediol (CAS 68855-18-5), Fatty acids, C8-18 and C18-unsatd., esters with neopentyl glycol (CAS 85186-86-3) and TMP ester of C8/C10 fatty (CAS 11138-60-6) are selected as source substances.
In vitro mutagenicity in bacteria
In summary, two reliable genetic toxicity studies addressing mutagenicity in bacterial cells in vitro are available.
CAS 97281-24-8
A key bacterial gene mutation study (Ames test) performed equivalent or similar to OECD guideline 471 and in compliance with GLP with Fatty acids, C8-10 (even numbered), diesters with neopentyl glycol and di- and triesters with trimethylolpropane (CAS 97281-24-8) is available (BASF, 1988d). The strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538 were tested according to the plate incorporation (experiment 1+2) procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). Both experiments were conducted in three repetitions at concentrations from 8 to 5000 µg/plate or vehicle (Tween 80/water) alone. There was no biologically relevant increase in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of metabolic activation. Appropriate solvent and positive controls were included into the test and gave the expected results. Hence, the test item was considered to be not mutagenic to bacteria under the conditions of the test.
CAS 85186-86-3
The mutagenic potential of Fatty acids, C8-18 and C18-unsatd., esters with neopentylglycol (CAS 85186-86-3) was tested in a reverse mutation assay performed comparable to OECD guideline 471 and under GLP conditions (Oleon, 2012). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA were used. Tester strains were incubated with test material concentrations of 50, 150, 500, 1500, and 5000 µg/plate dissolved in acetone with and without the addition of a metabolic activation system (phenobarbital and beta-naphthoflavone induced rat liver S9 mix). The first experiment was a plate incorporation assay and the repeat experiment was carried out using an additional 20 min pre-incubation. Vehicle, negative 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 controls was observed in all strains treated with the test substance, neither in the presence nor in the absence of metabolic activation. No cytotoxicity was observed in the plate incorporation assay. However, cytotoxicity was seen at 5000 µg/plate without S9 mix in the TA100, TA1535 and TA1537 strains in the pre-incubation assay. Thus, Fatty acids, C8-18 and C18-unsatd., esters with neopentylglycol did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested under the conditions of this test.
In vitro cytogenicity in mammalian cells
In summary, two reliable genetic toxicity studies addressing cytogenicity in mammalian cells in vitro are available.
CAS 68855-18-5
A chromosome aberration test was conducted with the test substance according to OECD guideline 473 and under GLP conditions in human lymphocytes (Croda, 2013a). Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatments conditions were used for the study. In experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9-mix), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9-mix) with a 20-hour expression period. In experiment 2, the 4 hours exposure with addition of S9-mix was repeated (using a 1% final S9-mix concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments, selected using data from the preliminary toxicity test, were 12.5, 25, 50, 100, 200, 400 µg/mL for all the four treatment conditions. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and the activity of the metabolising system. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either the absence or presence of S9-mix, in two separate experiments. In conclusions the test item was considered to be non-clastogenic to human lymphocytes in vitro.
CAS 11138-60-6
An in vitro mammalian chromosome aberration test was performed with Fatty acids, 8-10 (even numbered), di- and triesters with propylidynetrimethanol (CAS 11138-60-6) in Chinese Hamster Ovary cells (CHO) according to OECD guideline 473 and under GLP conditions (Exxon, 1986). Duplicate cultures of CHO cells were evaluated for chromosome aberrations in the presence and absence of metabolic activation (Arochlor 1254 induced rat liver S9-mix). Cells were exposed for 4 and 20 hours without and for 4 hours with metabolic activation. The test substance was dissolved in ethanol and used at concentrations of 625, 1250, 2500, 5000 µg/mL. Cytotoxicity was observed at the highest dose tested regardless of metabolic activation. Mitomycin C and cyclophosphamide was used as positive control without and with metabolic activation respectively. Vehicle (solvent) controls induced aberration frequencies within the range expected. Positive control material induced statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolizing system. Evaluation of 200 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations at any dose level tested in comparison to the negative controls. The test material was therefore considered to be non-clastogenic to CHO cells in vitro.
In vitro mutagenicity in mammalian cells
CAS 68855-18-5
A L5178Y mouse lymphoma assay was conducted according to OECD guideline 476 and under GLP conditions. Two independent experiments were performed (Croda, 2013b). In experiment 1, L51787Y TK +/- mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4 h exposure groups both in absence and presence of metabolic activation(2% S9-mix). In experiment 2, the cells were treated with the test item at eight dose levels using a 4 h exposure group in the presence of metabolic activation (1% S9-mix) and a 24 h exposure group in the absence of metabolic activation. The dose range of the test substance was selected following the results of a preliminary toxicity test and was determined to be 1.6 to 102.5 µg/mL in both the absence and presence of metabolic activation in experiment 1. In experiment 2 the dose range was 1.6 to 102.5 µg/mL in the absence of metabolic activation, and 3.2 to 205 µg/mL in the presence of metabolic activation. The maximum dose levels used in the test were limited by precipitate and test substance induced toxicity. A precipitate of the substance was observed at and above 102.5 µg/mL in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.
Conclusion for genetic toxicity
There is one study available addressing mutagenicity in bacterial cells in vitro for the target substance Fatty acids, C8-10 (even numbered), diesters with neopentyl glycol and di- and triesters with trimethylolpropane (CAS 97281-24-8), but no information on the fifth tester strain (Ames test) as well as on cytogenicity and mutagenicity in mammalian cells is available with the target substance. Therefore analogue read-across from source substances was applied from in vitro studies on mammalian cells, using three source substances. The results of the available in vitro studies on source substances did not provide evidence indicative for mutagenic potential. Based on the available data, and following the analogue approach Fatty acids, C8-10 (even numbered), diesters with neopentyl glycol and di- and triesters with trimethylolpropane (CAS 97281-24-8) is not expected to be mutagenic and clastogenic in vitro.
Justification for classification or non-classification
According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Fatty acids, C8-10 (even numbered), diesters with neopentyl glycol and di- and triesters with trimethylolpropane (CAS 97281-24-8), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.
Therefore, based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.
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