Registration Dossier
Registration Dossier
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EC number: 943-730-3 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 1538, TA 100 and TA 98, and E. coli WP2 uvrA
Read-across from structural analogue source substances Dipentaerythritol hexaesters with fatty acids, C5 and C9iso (CAS No. 647028-25-9), Hexanoic acid, 2-ethyl-, 2,2-bis [ [(2-ethyl-1-oxohexyl)oxy] methyl] -1,3-propanediyl ester (CAS No. 7299-99-2), Fatty acids, C5-9 tetraesters with pentaerythritol (CAS No. 67762-53-2), and Fatty acids, C5-9, hexaesters with dipentaerythritol (CAS No. 67762-52-1)
Chromosome aberration (OECD 473): negative in CHO cells with and without metabolic activation
Read-across from structural analogue source substances Dipentaerythritol hexaesters with fatty acids, C5 and C9iso (CAS No. 647028-25-9) and Hexanoic acid, 2-ethyl-, 2,2-bis [ [(2-ethyl-1-oxohexyl)oxy] methyl] -1,3-propanediyl ester (CAS No. 7299-99-2)
Gene mutation in mammalian cells (OECD 476): negative in mouse lymphoma L5178Y cells with and without metabolic activation
Read-across from structural analogue source substance Pentaerythritol tetravalerate (CAS No. 15834-04-5)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 8 Dec 1998 - 19 January 1999
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- EU MEthod B.14 of Commission Directive 92/69/EEC
- Qualifier:
- according to guideline
- Guideline:
- other: USA, EPA (TSCA) OPPTS harmonised guidelines
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- The Department of Health of the Government of the United Kingdom, UK
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- "his operon" (for S. typhimurium strains) and "trp operon" (for E.coli strains)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Preliminary toxicity study: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
First and second experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Untreated negative controls:
- yes
- Remarks:
- untreated control plates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Other: -S9: N-ethyl-N´-nitro-N-nitrosoguanidine (3, 5 and 2 µg/plate respectively) for TA100, TA1535 and WP2uvrA; 9-Aminoacridine (80 µg/plate) TA1537; 4-Nitroquinolone-1oxide (0.2 µg/plate) for TA98
- Untreated negative controls:
- yes
- Remarks:
- untreated control plates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: +S9: 2 aminoanthracene 1 µg/plate for TA 100, 2 µg/plate for TA 1535 and TA 1537, and 10 µg/plate for WP2uvrA; Benzo(a)pyrene (5 µg/plate) for TA98.
- 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
DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn
OTHER: a preliminary toxicity test was carried out in the strains TA100 and WP2uvrA. - Evaluation criteria:
- The test material may be considered positive in this test if the following criteria are met: the test item to be considered mutagenic should have induced, a reproducibile, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
- Statistics:
- Mean values and standard deviation were calculated. Dunnett’s method of linear regression was used.
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- oily precipitate was observed at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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
- Remarks:
- oily precipitate was observed at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: an oily precipitate was observed at 5000 µg/plate; however this did not prevent the scoring of revertant colonies.
RANGE-FINDING/SCREENING STUDIES: The dose range for the main test was determined in a preliminary toxicity assay where the test material was tested at the following doses: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test material was non-toxic to the strains of bacteria used (TA 100 and WP2uvrA-).
COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Furthermore positive control values were at least two times the respective vehicle control value for each strain. The historical control ranges are presented in Table 1. - Conclusions:
- Interpretation of results:
negative - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 12 Apr - 17 Dec 1998
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- too less information on test material are available
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon and trp operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- 33.3, 100, 333, 1000, 3330, and 5000 µg/plate of the test material (one experiment)
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol (plating aliquot: 50 µL)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2 aminoanthracene (2.5 or 25 µg/plate, S9+, TA100, TA1535, TA1537, WP2uvrA), benzo(a)pyrene (2.5 µg/plate, S9+, TA98)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide (2.0 µg/plate, S9-, TA100 and TA1535), 2-nitrofluorene (1.0 µg/plate, S9-,TA98), 4-nitroquinoline-N-oxide (1.0 µg/plate, S9-, WP2uvrA), ICR-191 (2.0 µg/plate, S9-, TA1537)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 52 ± 4 hr
NUMBER OF REPLICATIONS: triplicates (one experiment)
DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or by thinning or disappearance of the bacterial background lawn - Evaluation criteria:
- The test material was considered positive in this test system if the following criteria are met: for the test item to be considered mutagenic, at least two- (for tester strains TA98, TA 100, and WP2uverA) and/or three-fold (for tester strains TA 1535 and TA 1537) increases in mean revertants per plate over the mean reventants per plate of the appropriate vehicle control must be observed. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
- Statistics:
- Mean values and standard deviations were calculated
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results:
negative - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- no analytical purity reported
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (S. typhimurium) and trp operon (E. coli)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague Dawley rats treated i.p. with a single dose of 500 mg/kg bw Arochlor 1254
- Test concentrations with justification for top dose:
- Range-finding toxicity study (in TA 100 and WP2 uvrA): 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate, with and without metabolic activation
Main study (all strains): 33.3, 100, 333, 1000, 3330 and 5000 µg/plate, with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- -S9: 2-NF (1 µg/plate, TA 98); SA (2 µg/plate, TA 100 and TA 1535); ICR-191 (2 µg/plate, TA 1537); 4-NQO (1 µg/plate, WP2 uvrA); +S9: BP (2.5 µg/plate, TA 98); 2-AA (2.5-25 µg/plate, TA 100, TA 1535, TA 1537 and WP2 uvrA)
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- other: 2-aminoanthracene; ICR-191
- Remarks:
- 2-NF: 2-nitrofluorene; SA: sodium azide; 4-NQO: 4-nitroquinoline-N-oxide; BP: benzo(a)pyrene; 2-AA: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 52 ± 4 h
NUMBER OF REPLICATIONS: triplicates each in one experiment
DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn - Evaluation criteria:
- The results of the test were considered positive, if the following criteria were met:
- tester strains TA 98, TA 100 and WP2 uvrA: for a test article to be considered positive, it must produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.
- tester strains TA 1535 and TA 1537: for a test article to be considered positive, it must produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article. - Statistics:
- Mean values and standard deviations of revertants per plate were calculated.
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the absence of S9 mix, slight precipitation of the test substance was observed in all experiments at concentrations ≥ 100 µg/plate. In the absence of S9 mix, slight precipitates were noted at ≥ 333 µg/plate in the preliminary cytotoxicity study and at ≥ 1000 µg/plate in the main study.
RANGE-FINDING/SCREENING STUDIES: in a preliminary cytotoxicity study, the tester strains TA 100 and WP2 uvrA were treated with the test substance at concentrations ranging from 6.67 to 5000 µg/plate in the presence and absence of metabolic activation (S9 mix). No cytotoxicity was observed in these strains up to the limit dose of 5000 µg/plate, neither with nor without addition of S9 mix. - Conclusions:
- Interpretation of results:
negative - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone
- Test concentrations with justification for top dose:
- Dose-finding study:
-S9 mix: 0, 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains)
+S9 mix: 0, 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains)
Main study:
-S9 mix: 0, 78.1, 156, 313, 625, 1250, 2500 μg/plate (all strains)
+S9 mix: 0, 78.1, 156, 313, 625, 1250, 2500 μg/plate (all strains)
Re-study:
-S9 mix; 0, 78.1, 156, 313, 625, 1250, 2500 μg/plate (TA1535) - Vehicle / solvent:
- acetone
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: -S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2, for TA100, TA98, WP2 uvrA), sodium azide (NaN3, for TA1535) and 9-aminoacridine hydrochloride (9-AA, for TA1537) +S9 mix; 2-Aminoanthracene (2-AA, for all strains)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min, 37°C
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: reduction of background lawn
OTHER:
A re-test with TA1535 without S9 mix was performed to show reproducibility. - Evaluation criteria:
- The test substance was considered positive if the number of revertant colonies increased more than double compared to the number of revertant of the vehicle control as well as a dose-dependeny was found.
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- precipitation was observed at concentrations of ≥ 1250 µg/plate at the end of the exposure period
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- precipitation was observed at concentrations of ≥ 1250 µg/plate at the end of the exposure period
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- No increase in revertant colonies was observed in the test with either the non-activation method (-S9 mix) or the activation method (+S9 mix).
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: deposits of oily droplets were oberseved at concentrations including and higher than 1250 µg/plate
RANGE-FINDING/SCREENING STUDIES:
Since contamination was found in strain TA1535 without S9 mix, a retest with this strain was performed. However, no increase in number of revertants was found in any strain. - Conclusions:
- Interpretation of results:
negative - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 26 Mar - 11 May 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- thymidine kinase locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640 supplemented with 5% (v/v) heat-inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with a combination of phenobarbital and ß-naphthoflavone
- Test concentrations with justification for top dose:
- First experiment: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL (with and without metabolic activation (8%, v/v))
Second experiment: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL (with metabolic activation (12%, v/v)); 0.1, 1, 3, 10, 33, 100, 200, 250 µg/mL (without metabolic activation) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- in the absence of S9-mix Migrated to IUCLID6: 15 and 5 µg/mL for 3 and 24 h treatment period
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- in the presence of S9-mix Migrated to IUCLID6: 7.5 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in suspension
DURATION
- Exposure duration: cells were exposed to the test material for 3 h and 24 h
- Expression time (cells in growth medium): Cells in the final suspension after treatment were counted with the coulter particle counter. For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. Cells were plated for the determination of the cloning efficiency and mutation frequency. For the determination of the mutation frequency cells were plated and incubated for 11-12 d. After that, cells were stained for 2 h by adding 0.5 mg/mL MTT (Sigma) to each well. The plates were scored for cloning efficiency and mutation frequency with the naked eye or with the microscope.
SELECTION AGENT (mutation assays): RPMI 1640 supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth
- Evaluation criteria:
- Measurement of cytotoxicity by determining the relative cloning efficiency (survival) or relative total growth of the cultures is usually initiated after the treatment period.
There are several criteria for determining a positive result, such as a concentration-related, or a reproducible increase in mutant frequency. - Statistics:
- The cloning efficiency (CE) was determined as follows:
P(0)= Number of empty wells divided by the total number of wells
CE= P(0)/number of cells plated per well
Relative survival rate (RS): RS= [CE(test)/CE(control)] x 100
Relative total growth (RTG): RTG= RSG x RSday2 / 100
Suspension growth (SG): [Day 0 cell count/1.25x10E005] x [Day 1 cell count/1.25x10E005] x [Day 2 cell count]
Relative suspension growth (RSG): SG(test)/SG(control) x 100
RSday2= CEday2(test) / CEday2(control) x 100
The growth rate (GR) was calculated for the solvent control cultures:
- 3 h treatment: [Day 1 cell count/1.25x105] x [Day 2 cell count/1.25x10E005]
- 24 h treatment: [Day 0 cell count/1.25x105] x [Day 1 cell count/1.25x10E005] x [Day 2 cell count/1.25x10E005]
The mutation frequency was expressed as the number of mutants per 106 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:
MF= {-ln P(0)/number of cells plated per well)/CE day2 x 10E-006
Small and large colony mutation frequencies were calculated in an identical manner. - Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- and above (precipitating concentration: 100 µg/mL, tested up to 250 µg/mL)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 100 µg/mL
RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity data was obtained by treating cells for 3 h and 24 h, respectively, with a number of increasing test substance concentrations. The highest concentration tested was 200 µg/ml due to poor solubility of the test substance. No toxicity was observed with and without metabolic activation up to and at the maximum dose level tested with 3 h incubation. 24 h incubation resulted in 64% relative suspension growth in the absence of metabolic activation.
COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all controls were in the range of the historical controls. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 8 Feb - 15 Apr 1999
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted in 1997
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes
- Remarks:
- The Department of Health of the Government of the United Kingdom, UK
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
Eagle’s MEM medium supplemented with:
-sodium bicarbonate
-HEPES buffer
-L-glutamine
-penicillin/streptomycin
-amphotericin B
-15% foetal calf serum (FCS) - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- First experiment:
4(20) h without and with S9: 39.06; 78.13; 156.25; 312.5; 625; 1250; 2500; 5000 µg/mL
Second experiment:
20 (20) h without S9: 156.25; 312.5; 625; 1250; 2500 and 5000 µg/mL
4(20) h with S9: 156.25; 312.5; 625; 1250; 2500 and 5000 µg/mL - 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:
- other: cyclophosphamide, 25 µg/mL in phopsphate buffered saline, +S9; ethyl methanesulphonate , 750 µg/mL and 500 µg/mL (Experiment 1 and 2 respectively), -S9 in dimethyl sulphoxide
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h (experiment 1, with and without S9); 4 and 20 h (experiment 2, with and without S9, respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h
SPINDLE INHIBITOR (cytogenetic assays): colcemid 0.1 µg/mL
STAIN (for cytogenetic assays): Gurrs Giemsa R66 5%
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 cells
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 increases 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 (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test or Chi-squared test.
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- 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
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: there was no observable change in pH
- Effects of osmolality: the osmolality did not increase by more than 50 mOSM
- Precipitation: a cloudy appearance of the test material was noted at all dose levels in both treatment groups, after four hours exposure
COMPARISON WITH HISTORICAL CONTROL DATA: Yes
ADDITIONAL INFORMATION ON CYTOTOXICITY: in Experiment 1 and 2 no dose-related toxicity was observed, a 50% mitotic inhibition was not achieved and the precipitate of the test item had no effect on the toxicity response curve. Thus, 1250, 2500 and 5000 µg/mL dose levels were selected for chromosome analysis. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- other: Chinese hamster lung (CHL/IU) cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Liquid medium Eagle-MEM (Asahi Techno Glass) supplemented with 10% inactivated (56 ℃, 30 min) calf serum (Invitrogen)
- Properly maintained: yes - 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:
- Pretest:
-S9 mix (short-term treatment): 0, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/mL
+S9 mix (short-term treatment): 0, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/mL
-S9 mix (continuous treatment): 0, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/mL
Main test:
-S9 mix (short-term treatment): 0, 1250, 2500, 5000 μg/mL
+S9 mix (short-term treatment): 0, 1250, 2500, 5000 μg/mL
-S9 mix (continuous treatment): 0, 1250, 2500, 5000 μg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: as the test material is not soluble in water - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: -S9 mix: Mitomycin C (0.1 µg/mL for short term treatment and 0.05 µg/mL for continuous treatment); +S9 mix: Cyclophosphamide (12.5 µg/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium (Pre- and Main test)
DURATION
- Exposure duration: 6 h (short-term treatment), 24 h (continuous treatment);
- Expression time (cells in growth medium): 18 h (short-term treatment)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h (short-term and continuous treatment)
SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.2 µg/mL) for 2 h
STAIN (for cytogenetic assays): 1.2 vol% Giemsa for 12 min
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 200/dose
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- Significant difference of the substance-induced frequency of structural chromosome aberrations was considered positive if a dose-dependency or reproducibility was observed in the treated group compared with the vehicle control group.
- Statistics:
- The significance level of the frequency of polyploid cells or cells with an abnormal structure of each test group was tested using a direct probability calculation method of Fisher (2.5% one-sided significance level). For dose dependence the trend test according to Cochran Armitage (2.5% one-sided significance level) was used.
- Species / strain:
- other: Chinese hamster lung (CHL/IU) cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: visible precipitation was observed at the end of the expoure period (short-term and continous treatment)
RANGE-FINDING/SCREENING STUDIES:
In the pretest, visible precipitation was observed at the end of exposure period at concentrations including and higher than 625 µg/mL. However, no inhibition of cell growth could be detected in any concentrations with and without S9 mix after short-term and continuous exposure. - Conclusions:
- Interpretation of results: negative
Referenceopen allclose all
Table 1: Historical control ranges
History profile-Vehicle control values |
|
||||||
-S9 |
TA 100 |
TA 98 |
TA 1535 |
TA 1538 |
TA 1537 |
WP2uvrA |
TA 102 |
Extreme values |
61-196 |
13-56 |
10-39 |
6-41 |
4-17 |
12-39 |
216-339 |
Mean |
117± 24.7 |
28± 6.7 |
24± 5.2 |
21± 6.8 |
10± 2.0 |
20± 4.6 |
264 29.8 |
History profile-Vehicle control values |
|||||||
+S9 |
TA 100 |
TA 98 |
TA 1535 |
TA 1538 |
TA 1537 |
WP2uvrA |
TA 102 |
Extreme values |
63-177 |
14-52 |
11-39 |
11-50 |
5-20 |
11-40 |
205-343 |
Mean |
120± 22.2 |
33± 7.1 |
18± 4.0 |
25± 6.2 |
11± 2.3 |
21± 4.8 |
283± 32.7 |
History profile-Positive control values |
|||||||
-S9 |
TA 100 |
TA 98 |
TA 1535 |
TA 1538 |
TA 1537 |
WP2uvrA |
TA 102 |
Extreme values |
277-1126 |
127-698 |
163-1005 |
190-799 |
161-1149 |
342-1209 |
540-1188 |
Mean |
613±175.7 |
203±57.2 |
466±208.4 |
484±146.0 |
812±201.3 |
834±197.2 |
785±172.1 |
+S9 |
TA 100 |
TA 98 |
TA 1535 |
TA 1538 |
TA 1537 |
WP2uvrA |
TA 102 |
Extreme values |
412-1315 |
198-757 |
139-516 |
212-915 |
123-718 |
225-1089 |
511-1090 |
Mean |
949± 189.7 |
420± 112.5 |
291± 63.6 |
454± 131.0 |
266± 90.2 |
734± 210.5 |
705± 126.6 |
Table 2: 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 |
WP2uvr A- |
TA98 |
TA1537 |
||
– |
0 |
81 ± 11.0 |
16 ± 1.5 |
25 ± 3.5 |
19 ± 2.5 |
7 ± 3.8 |
– |
50 |
77 ± 2.1 |
14 ± 0.6 |
22 ± 0.6 |
24 ± 0.6 |
9 ± 3.6 |
– |
150 |
80 ± 12.2 |
14 ± 1.0 |
22 ±0.6 |
20 ± 6.7 |
10 ± 3.8 |
– |
500 |
83 ± 10.8 |
13 ± 2.3 |
23 ± 2.9 |
21 ± 6.0 |
7 ± 2.6 |
– |
1500 |
76 ± 1.5 |
18 ± 5.2 |
18 ± 2.6 |
22 ± 1.2 |
7 ± 3.2 |
– |
5000 |
90 ± 11.8 P |
18 ± 8.2 P |
24 ± 4.0 P |
28 ± 4.9 P |
5 ± 0.6 P |
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) |
323 ± 11.7 |
283 ± 10.0 |
503 ± 24.2 |
166 ± 5.3 |
1027 ± 358.2 |
|
|
||||||
|
TA 100 |
TA1535 |
WP2uvr A- |
TA98 |
TA1537 |
|
+ |
0 |
105 ± 7.8 |
11 ± 1.2 |
28 ± 3.8 |
30 ± 3.1 |
20 ± 3.6 |
+ |
50 |
101 ± 7.0 |
12 ± 0.6 |
27 ± 3.8 |
29 ± 6.6 |
20 ± 3.2 |
+ |
150 |
102 ± 5.5 |
16 ± 4.0 |
32 ± 1.5 |
27 ± 4.0 |
23 ± 2.0 |
+ |
500 |
93 ± 16.6 |
14 ± 4.0 |
28 ± 6.6 |
30 ± 6.7 |
20 ± 1.2 |
+ |
1500 |
86 ± 3.6 |
15 ± 3.5 |
26 ± 4.0 |
31 ± 3.1 |
17 ± 6.5 |
+ |
5000 |
93 ± 7.8 P |
12 ± 1.2 P |
24 ± 1.5 P |
33 ± 5.7 P |
15 ± 2.6 P |
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) |
920 ± 153.0 |
198 ± 9.3 |
555 ± 27.8 |
551 ± 132.0 |
196 ± 6.5 |
|
Table 3: 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 ± SD) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2uvr A- |
TA98 |
TA1537 |
||
– |
0 |
96 ± 4.5 |
18 ± 4.2 |
26 ± 3.8 |
18 ± 5.3 |
11 ± 6.5 |
– |
50 |
97 ± 18.6 |
17 ± 4.0 |
29 ± 6.5 |
16 ± 3.6 |
12 ± 0.6 |
– |
150 |
100 ± 9.5 |
20 ± 9.0 |
28 ±0.0 |
16 ± 3.8 |
11 ± 1.7 |
– |
500 |
101 ± 8.4 |
18 ± 6.2 |
33 ± 6.0 |
19± 4.4 |
12 ± 1.2 |
– |
1500 |
87 ± 6.4 |
17 ± 7.1 |
29 ± 7.1 |
22 ± 0.0 |
12 ± 1.2 |
– |
5000 |
93 ± 1.0 P |
19 ± 3.1 P |
33 ± 5.1 P |
17 ± 3.2 P |
15 ± 6.0 P |
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) |
880 ± 31.5 |
257 ± 34.4 |
1191 ± 59.3 |
166 ± 4.6 |
1084 ± 263.4 |
|
|
||||||
|
TA 100 |
TA1535 |
WP2uvr A- |
TA98 |
TA1537 |
|
+ |
0 |
85 ± 4.0 |
14 ± 3.5 |
35± 1.2 |
31 ± 5.0 |
21 ± 1.5 |
+ |
50 |
91 ± 7.5 |
19 ± 0.7 |
35 ± 10.1 |
30 ± 8.0 |
24 ± 1.5 |
+ |
150 |
84 ± 9.5 |
14 ± 1.0 |
34 ± 6.1 |
29 ± 10.7 |
22 ± 3.5 |
+ |
500 |
101 ± 14.3 |
10 ± 0.6 |
42 ± 0.6 |
28 ± 4.5 |
22 ± 2.1 |
+ |
1500 |
94 ± 8.7 |
14 ± 0.6 |
36 ± 5.3 |
31 ± 4.7 |
21 ± 1.0 |
+ |
5000 |
101 ± 6.7 P |
14 ± 3.2 P |
34 ± 7.0 P |
30 ± 5.5 P |
22 ± 1.2 P |
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) |
865 ± 43.2 |
246 ± 7.5 |
1051 ± 90.0 |
611 ± 51.5 |
182 ± 7.0 |
|
ENNG = N-Ethyl-N´-nitro-N-nitrosoguanidine
4NQO = 4-Nitroquinoline-1-oxide
9AA = 9-Aminoacridine
BP = Benzo(a)pyrene
2AA = 2-Aminoanthracence
P = precipitate
Table 1. Test results of experiment (plate incorporation)
Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD) |
|||||
EXPERIMENT |
|||||
S9-Mix |
Without
|
||||
Concentration (per plate) |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
SC |
25 ± 2 |
80 ± 9 |
14 ± 8 |
3 ± 1 |
26 ± 3 |
Test material |
|
||||
33.3 µg |
19 ± 4 |
88 ± 5 |
12 ± 5 |
4 ± 4 |
19 ± 2 |
100 µg |
21 ± 7 |
96 ± 12 |
11 ± 2 |
5 ± 4 |
19 ± 4 |
333 µg |
22 ± 2 |
92 ± 9 |
13 ± 2 |
5 ± 5 |
23 ± 10 |
1000 µg |
25 ± 6 |
97 ± 11 |
25 ± 2 |
5 ± 3 |
30 ± 3 |
3330 µg |
22 ± 1 |
101 ± 3 |
10 ± 1 |
3 ± 2 |
32 ± 2 |
5000 µg |
20 ± 6 |
85 ± 6 |
15 ± 3 |
4 ± 2 |
27 ± 3 |
PC |
|
||||
2-NF |
206 ± 42 |
- |
- |
- |
- |
SA |
- |
549 ± 71 |
480 ± 3 |
- |
- |
ICR-191 |
- |
- |
- |
277 ± 33 |
- |
4-NQO |
- |
- |
- |
- |
260 ± 43 |
S9-Mix
|
With |
||||
Concentration (per plate) |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
SC |
36 ± 2 |
102 ± 2 |
19 ± 1 |
7 ± 1 |
25 ± 5 |
Test material |
|
||||
33.3 µg |
36 ± 7 |
93 ± 9 |
15 ± 2 |
8 ± 4 |
22 ± 5 |
100 µg |
34 ± 7 |
97 ± 17 |
16 ± 4 |
9 ± 2 |
22 ± 6 |
333 µg |
34 ± 6 |
87 ± 6 |
17 ± 2 |
9 ± 4 |
27 ± 3 |
1000 µg |
39 ± 9 |
94 ± 17 |
17 ± 3 |
8 ± 2 |
26 ± 6 |
3330 µg |
38 ± 8 |
92 ± 6 |
16 ± 4 |
6 ± 3 |
29 ± 11 |
5000 µg |
34 ± 5 |
139 ± 5 |
20 ± 3 |
3 ± 2 |
24 ± 4 |
PC |
|
|
|
|
|
BP |
471 ± 14 |
- |
- |
- |
- |
2-AA |
- |
918 ± 296 |
124 ± 6 |
171 ± 32 |
278 ± 24 |
SC = Solvent control; PC = Positive control substances; SD = standard deviation; 2-NF: 2-nitrofluorene; SA: sodium azide; 4-NQO: 4-nitroquinoline-N-oxide; BP: benzo(a)pyrene; 2-AA: 2-aminoanthracene |
|||||
Pretest:
With or without S9-Mix |
Test substance concentration |
Mean number of revertant colonies per plate |
|||||
(μg/plate) |
(average of 3 plates ± Standard deviation) |
||||||
|
Base-pair substitution type |
Frameshift type |
|||||
|
TA 100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
– |
0 |
120 ± 10 |
- |
25 ± 3 |
22 ± 1 |
7 ± 3 |
|
– |
8.19 |
18 ± 6 |
- |
24 ± 3 |
22 ± 2 |
5 ± 1 |
|
– |
20.5 |
126 ± 7 |
- |
24 ± 3 |
23 ± 4 |
7 ± 3 |
|
– |
51.2 |
110 ± 4 |
- |
26 ± 4 |
30 ± 2 |
7 ± 1 |
|
– |
128 |
117 ± 8 |
- |
30 ± 2 |
26 ± 3 |
7 ± 2 |
|
– |
320 |
117 ± 9 |
- |
23 ± 2 |
23 ± 3 |
5 ± 2 |
|
– |
800 |
118 ± 9 |
- |
22 ± 3 |
25 ± 3 |
7 ± 3 |
|
– |
2000 + |
113 ± 4 |
- |
28 ± 3 |
28 ± 4 |
8 ± 3 |
|
– |
5000 + |
118 ± 3 |
|
24 ± 3 |
25 ± 3 |
8 ± 2 |
|
Positive controls, –S9 |
Name |
AF-2 |
- |
AF-2 |
AF-2 |
9-AA |
|
Concentrations (μg/plate) |
0.01 |
- |
0.01 |
0.1 |
80 |
||
Mean No. of colonies/plate (average of 3 ± SD) |
590 ± 25 |
- |
151 ± 9 |
706 ± 25 |
197 ± 25 |
||
+ |
0 |
121 ± 8 |
12 ± 2 |
24 ± 2 |
28 ± 5 |
14 ± 1 |
|
+ |
8.19 |
132 ± 6 |
10 ± 2 |
22 ± 2 |
34 ± 3 |
12 ± 2 |
|
+ |
20.5 |
124 ± 7 |
10 ± 2 |
28 ± 3 |
33 v 3 |
14 ± 3 |
|
+ |
51.2 |
127 ± 2 |
12 ± 2 |
30 ± 4 |
35 ± 1 |
12 ± 3 |
|
+ |
128 |
121 ± 4 |
12 ± 2 |
25 ± 3 |
30 ± 3 |
11 ± 1 |
|
+ |
320 |
126 ± 11 |
12 ± 3 |
25 ± 4 |
28 ± 3 |
12 ± 2 |
|
+ |
800 |
139 ± 11 |
11 ± 1 |
25 ± 2 |
28 ± 4 |
12 ± 2 |
|
+ |
2000 + |
132 ± 4 |
11 ± 1 |
28 ± 5 |
31 ± 3 |
13 ± 2 |
|
+ |
5000 + |
134 ± 7 |
9 ± 3 |
31 ± 2 |
30 ± 3 |
14 ± 2 |
|
Positive controls, +S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
|
Concentrations (μg/plate) |
1 |
2 |
10 |
0.5 |
2 |
||
Mean No. of colonies/plate (average of 3 ± SD) |
1044 ± 114 |
304 ± 24 |
682 ± 17 |
359 ± 13 |
135 ± 12 |
||
+: precipitation
AF-2:2 -(2 -furyl)-3 -(5 -nitro-2 -furyl) acrylamide
NaN3: Sodium azide
9 -AA:9 - aminoacridine hydrochloride
2 -AA.2 - aminoanthracene
Main test:
With or without S9-Mix |
Test substance concentration |
Mean number of revertant colonies per plate |
|||||
(μg/plate) |
(average of 3 plates ± Standard deviation) |
||||||
|
Base-pair substitution type |
Frameshift type |
|||||
|
TA 100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
– |
0 |
119 ± 5 |
10 ± 1 |
24 ± 3 |
20 v 1 |
7 ± 1 |
|
– |
78.1 |
126 ± 12 |
9 ± 3 |
27 ± 3 |
19 ± 3 |
9 ± 4 |
|
– |
156 |
120 ± 11 |
14 ± 1 |
24 ± 2 |
22 ± 2 |
8 ± 1 |
|
– |
313 |
123 ± 7 |
13 ± 1 |
23 ± 3 |
17 ± 3 |
6 ± 1 |
|
– |
625 |
122 ± 16 |
11 ± 3 |
23 ± 2 |
17 ± 2 |
7 ± 1 |
|
– |
1250 + |
112 ± 3 |
13 ± 3 |
26 ± 3 |
24 ± 6 |
7 ± 1 |
|
– |
2500 + |
128 ± 9 |
11 ± 2 |
27 ± 4 |
22 ± 3 |
9 v 3 |
|
Positive controls, –S9 |
Name |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
9-AA |
|
Concentrations (μg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
80 |
||
Mean No. of colonies/plate (average of 3 ± SD) |
741 ± 57 |
436 ± 25 |
152 ± 12 |
689 ± 53 |
205 ± 6 |
||
+ |
0 |
131 ± 5 |
11 ± 2 |
29 ± 8 |
30 ± 5 |
12 ± 2 |
|
+ |
78.1 |
132 ± 13 |
12 ± 1 |
27 ± 9 |
30 ± 4 |
13 ± 4 |
|
+ |
156 |
145 ± 5 |
12 ± 1 |
24 ± 4 |
34 ± 5 |
10 ± 3 |
|
+ |
313 |
131 ± 3 |
13 ± 2 |
26 ± 6 |
29 ± 2 |
16 ± 5 |
|
+ |
625 |
148 ± 6 |
12 ± 2 |
24 ± 3 |
26 ± 1 |
13 ± 3 |
|
+ |
1250 + |
135 ± 145 |
10 ± 1 |
27 ± 4 |
28 ± 3 |
11 ± 3 |
|
+ |
2500 + |
152 ± 3 |
10 ± 2 |
31 ± 4 |
28 ± 3 |
14 ± 4 |
|
Positive controls, +S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
|
|
Concentrations (μg/plate) |
1 |
2 |
10 |
0.5 |
2 |
||
Mean No. of colonies/plate (average of 3 ± SD) |
1047 ± 53 |
362 ± 6 |
720 ± 49 |
343 ± 45 |
129 ± 13 |
||
+: precipitation
AF-2: 2 -(2 -furyl)-3 -(5 -nitro-2 -furyl) acrylamide
NaN3: Sodium azide
9 -AA: 9 - aminoacridine hydrochloride
2 -AA. 2 - aminoanthracene
Re-test:
With or without S9-Mix |
Test substance concentration |
Mean number of revertant colonies per plate |
|
(μg/plate) |
(average of 3 plates ± Standard deviation) |
||
|
Base-pair substitution type |
||
|
TA1535 |
||
– |
0 |
9 ± 2 |
|
– |
78.1 |
8 ± 1 |
|
– |
156 |
13 ± 2 |
|
– |
313 |
13 ± 1 |
|
– |
625 |
11 ± 3 |
|
– |
1250 |
10 ± 1 |
|
– |
2500 |
10 ± 3 |
|
Positive controls, –S9 |
Name |
NaN3 |
|
Concentrations (μg/plate) |
0.5 |
||
Mean No. of colonies/plate (average of 3 ± SD) |
523 ± 20 |
NaN3: Sodium azide
Table 1: Experiment 1 - 3 hours with and without S9 mix
Dose (µg/ml) |
RSG (%) |
CE day2 (%) |
RS day2 (%) |
RTG (%) |
mutation frequency x 10-6 |
|
|
|
|
|
total |
Without metabolic activation, 3 h treatment |
|||||
SC1 |
100 |
94 |
100 |
100 |
89 |
SC2 |
108 |
73 |
|||
0.03 |
98 |
101 |
100 |
98 |
63 |
0.1 |
92 |
99 |
98 |
90 |
83 |
0.3 |
111 |
102 |
101 |
112 |
58 |
1 |
107 |
98 |
97 |
104 |
64 |
3 |
110 |
101 |
100 |
110 |
83 |
10 |
98 |
99 |
98 |
96 |
83 |
33 |
98 |
110 |
109 |
106 |
90 |
100* |
74 |
94 |
93 |
69 |
97 |
MMS |
70 |
63 |
63 |
44 |
1022 |
With 8% (v/v) metabolic activation, 3 h treatment |
|||||
SC1 |
100 |
77 |
100 |
100 |
82 |
SC2 |
84 |
87 |
|||
0.03 |
96 |
90 |
112 |
107 |
71 |
0.1 |
92 |
104 |
129 |
119 |
60 |
0.3 |
80 |
108 |
135 |
108 |
55 |
1 |
93 |
105 |
131 |
121 |
69 |
3 |
97 |
90 |
112 |
109 |
65 |
10 |
95 |
84 |
104 |
99 |
71 |
33 |
93 |
81 |
101 |
94 |
91 |
100* |
42 |
83 |
103 |
43 |
98 |
CP |
20 |
37 |
47 |
9 |
1107 |
Table 2: Experiment 2 - 3 hours with and 24 hours without S9 mix
Dose (µg/ml) |
RSG (%) |
CE day2 (%) |
RS day2 (%) |
RTG (%) |
mutation frequency x 10-6 |
|
|
|
|
|
total |
Without metabolic activation, 24 h treatment |
|||||
SC1 |
100 |
102 |
100 |
100 |
62 |
SC2 |
104 |
57 |
|||
0.1 |
97 |
83 |
80 |
78 |
87 |
1 |
94 |
105 |
102 |
96 |
68 |
3 |
102 |
90 |
87 |
89 |
65 |
10 |
104 |
115 |
111 |
115 |
54 |
33 |
105 |
83 |
80 |
84 |
53 |
100* |
102 |
98 |
95 |
97 |
55 |
200* |
116 |
104 |
101 |
116 |
52 |
250* |
112 |
108 |
105 |
118 |
51 |
MMS |
80 |
81 |
79 |
63 |
631 |
With 12% (v/v) metabolic activation, 3 h treatment |
|||||
SC1 |
100 |
77 |
100 |
100 |
60 |
SC2 |
91 |
84 |
|||
0.03 |
116 |
58 |
69 |
81 |
108 |
0.1 |
97 |
80 |
95 |
93 |
86 |
0.3 |
94 |
80 |
95 |
90 |
76 |
1 |
99 |
81 |
97 |
96 |
88 |
3 |
102 |
89 |
106 |
108 |
71 |
10 |
104 |
86 |
103 |
106 |
73 |
33 |
119 |
86 |
103 |
122 |
83 |
100* |
105 |
77 |
91 |
96 |
72 |
CP |
31 |
54 |
64 |
20 |
814 |
RSG: Relative Suspension Growth; CE: Cloning efficiency; RS: Relative Survival; RTG: Relative Total Growth; SC: Solvent Control (DMSO); MMS: Methylmethansulfonate; CP: Cyclophosphamide
*: Precipitation of test substance
Table 1: Test results of experiment 1
Test item |
Concentration |
Mitotic Index |
Aberrant cells in % |
|
|
in µg/mL |
in % |
with gaps |
without gaps |
Exposure period 4 h, fixation time 20 h, without S9 mix |
||||
Acetone |
0 |
100 |
3.0 |
2.5 |
EMS |
750 |
50 |
20 |
12.5 |
Test substance |
1250 |
78 |
3.0 |
2.0 |
2500 |
83 |
5.5 |
1.0 |
|
5000 |
70 |
2.0 |
1.5 |
|
Exposure period 4 h, fixation time 20 h, with S9 mix |
||||
Acetone |
0 |
100 |
3.5 |
2.0 |
CP |
25 |
29 |
14.5 |
9.0 |
Test substance |
1250 |
87 |
3.0 |
3.0 |
2500 |
125 |
3.5 |
0.5 |
|
5000 |
117 |
1.5 |
1.0 |
|
EMS: ethyl methanesulphonate;
CP: Cyclophosphamide (positive controls)
Table 2: Test results of experiment 2
Test item |
Concentration |
Mitotic Index |
Aberrant cells in % |
|
|
in µg/mL |
in % |
with gaps |
without gaps |
Exposure period 20 h, fixation time 20 h, without S9 mix |
||||
Acetone |
0 |
100 |
1.5 |
0.5 |
EMS |
750 |
58 |
36.7 |
20.0 |
Test substance |
1250 |
123 |
2.0 |
0.5 |
2500 |
110 |
0.5 |
0.0 |
|
5000 |
99 |
2.0 |
0.0 |
|
Exposure period 4 h, fixation time 20 h, with S9 mix |
||||
Acetone |
0 |
100 |
2.0 |
0.5 |
CP |
25 |
25 |
19.0 |
11.5 |
Test substance |
1250 |
89 |
1.5 |
0.0 |
2500 |
89 |
1.0 |
0.0 |
|
5000 |
112 |
1.0 |
0.0 |
|
EMS: ethyl methanesulphonate;
CP: Cyclophosphamide (positive controls)
Results:
Short term treatment; without S9-mix |
|||||
Compound |
Dose (µg/ml) |
Time of exposure (h) |
Relative cell growth (%) |
No. of cells analyzed |
No. of cells with aberrations -gap (%) |
test substance |
0 |
6 |
100 |
200 |
0.5 |
test substance |
1250* |
6 |
71.9 |
200 |
1 |
test substance |
2500* |
6 |
70.2 |
200 |
0.5 |
test substance |
5000* |
6 |
64.8 |
200 |
0 |
positive control MMC |
0.1 |
6 |
90.7 |
200 |
48.5 |
Short term treatment; with S9-mix |
|||||
Compound |
Dose (µg/ml) |
Time of exposure (h) |
Relative cell growth (%) |
No. of cells analyzed |
No. of cells with aberrations -gap (%) |
test substance |
0 |
6 |
100 |
200 |
0 |
test substance |
1250* |
6 |
73.7 |
200 |
0.5 |
test substance |
2500* |
6 |
68.1 |
200 |
1 |
test substance |
5000* |
6 |
69.1 |
200 |
0 |
positive control CP |
12.5 |
6 |
83.1 |
200 |
34 |
Continuous treatment; without S9 -mix |
|||||
Compound |
Dose (µg/ml) |
Time of exposure (h) |
Relative cell growth (%) |
No. of cells analyzed |
No. of cells with aberrations -gap (%) |
test substance |
0 |
24 |
100 |
200 |
0.5 |
test substance |
1250* |
24 |
78.9 |
200 |
0.5 |
test substance |
2500* |
24 |
64.9 |
200 |
0 |
test substance |
5000* |
24 |
64.9 |
200 |
0.5 |
positive control MMC |
0.05 |
24 |
91.2 |
200 |
38 |
*: precipitation was observed at the end of exposure period
CP: Cyclophosphamide
MMC: Mitomycin C
No increase in chromosomal aberrations was observed with either the short-term treatment (-S9 mix and +S9 mix) or the continuous treatment.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Mammalian erythrocyte micronucleus formation in vivo (OECD 474): negative after intraperitoneal and oral application
Read-across from structural analogue source substance Pentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS No. 68424-31-7)
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 May - 08 July 1992
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- adopted in 1983
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK Limited), Margate, Kent, UK
- Age at study initiation: 5-9 weeks for phase I (determination of the maximum tolerated dose) and 7-9 weeks for phase II (Micronucleus test) of the study
- Assigned to test groups randomly: Yes
- Housing: 5 per cage in mobile mouse cage racks, housed per sex
- Diet: Porton Combined Diet, ad libitum
- Water: filtered tap water, ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): 25
- Photoperiod (hrs dark / hrs light): 12 / 12 - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg - Details on exposure:
- The study consisted in two phases: in phase I the maximum tolerated dose (MTD) was determined, on the basis of lethalities or severe toxicity observed over a four-day observation period following a single intraperitoneal injection.
In phase II, male and female animals were weighed and given a single intraperitoneal injection of corn oil (vehicle control), cyclophosphamide (positive control) or test substance prepared in corn oil. - Duration of treatment / exposure:
- Single dose
- Frequency of treatment:
- Single dose
- Post exposure period:
- 24 h and 48 h
- Dose / conc.:
- 5 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 65 mg/kg bw in physiological saline - Tissues and cell types examined:
- Monochromatic and polychromatic erythrocytes
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
No deaths or severe adverse effects occurred in Phase I of the study with doses up to 5000 mg/kg bw. This dose was selected as MTD.
TREATMENT AND SAMPLING TIMES: 24 h and 48 h after dosing
DETAILS OF SLIDE PREPARATION: Bone Marrow smears were stained with polychrome methylene blue and eosin
METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were evaluated for micronuclei per slide. In addition, 1000 erythrocytes were counted to determine the percentage of polychromatic erythrocytes in the total erythrocyte population. - Evaluation criteria:
- Increase in the incidence of micronucleated polychromatic erythrocytes in any sex or at any time point.
Percentage of polychromatic erythrocytes. - Statistics:
- The incidence of micronucleated polychromatic erythrocytes and percentage of polychromatic erythrocytes in the erythrocyte sample were considered by analysis of variance regarding each combination of sampling time, dose level and sex as a separate group. Results were examined to determine wether any differences between vehicle control and test substance treated groups were consistent between sexes and across sampling times.
Each group mean was compared with the vehicle control group mean at the corresponding sampling time using a one-sided Student´s t-test based on the error mean square in the analysis. - Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes over the vehicle control values were seen in either sex at either of the sampling times.
Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the female animals treated with the vehicle control or with the test material. A small, but significant decrease was, however, noted in male mice treated with the test material at 5000 mg/kg bw. This small decrease is, however, considered not to be statistically significant compared to the concurrent control values.
The positive control induced stastistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen. - Conclusions:
- Interpretation of results: negative
Reference
Mean incidence of micronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes ± Standard Deviation at two sampling times. n=5
Table 1: Males
Group |
Compound |
Dose |
Mean Incidence |
|
24 h |
48 h |
|||
11 |
Vehicle control (corn oil) |
10 mL/kg |
0.8 ± 0.8 |
1.0 ± 1.2 |
12 |
Cyclophosphamide |
65 mg/kg |
24.4 ± 6.0** |
|
13 |
Test substance |
5000 mg/kg |
0.6 ± 0.6 |
0.4 ± 0.6 |
Table 2: Females
Group |
Compound |
Dose |
Mean Incidence |
|
24 h |
48 h |
|||
11 |
Vehicle control (corn oil) |
10 ml/kg |
0.2 ± 0.5 |
1.4 ± 1.1 |
12 |
Cyclophosphamide |
65 mg/kg |
18.4 ± 7.3** |
|
13 |
Test substance |
5000 mg/kg |
0.4 ± 0.9 |
0.4 ± 0.9 |
Mean percentage of polychromatic erythrocytes ± Standard Deviation at two sampling times. n=5
Table 3: Males
Group |
Compound |
Dose |
Mean Incidence |
|
24 h |
48 h |
|||
11 |
Vehicle control (corn oil) |
10 ml/kg |
48.0 ± 5.6 |
44.3 ± 7.5 |
12 |
Cyclophosphamide |
65 mg/kg |
41.4 ± 4.4* |
|
13 |
Test substance |
5000 mg/kg |
42.2 ± 7.0* |
43.3 ± 1.9 |
Table 4: Female
Group |
Compound |
Dose |
Mean Incidence |
|
24 h |
48 h |
|||
11 |
Vehicle control (corn oil) |
10 ml/kg |
41.9 ± 4.8 |
41.9 ± 1.7 |
12 |
Cyclophosphamide |
65 mg/kg |
45.9 ± 3.49 |
|
13 |
Test substance |
5000 mg/kg |
46.5 ± 5.8 |
48.0 ± 5.2 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for grouping of substances and read-across
The read-across from analogue source substances approach comprises aliphatic esters of poly-functional alcohols containing two to six reactive hydroxyl groups and one to six fatty acid chains. The analogue approach contains mono constituent, multi-constituent and UVCB substances with fatty acid carbon chain lengths ranging from C5 - C28, which are mainly saturated but also mono unsaturated C16 and C18, polyunsaturated C18, branched C5 and C9, branched C14 - C22 building mono-, di-, tri-, and tetra esters with an alcohol (i.e. the polyol).
The available data allows for an accurate hazard and risk assessment of the target substance and the read-across concept is applied for the assessment of environmental fate and environmental and human health hazards. Thus, where applicable, environmental and human health effects are predicted from adequate and reliable data for source substances within the group by interpolation to the target substance applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No. 1907/2006. In particular, for each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.
A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 7.1 and 13).
In vitro gene mutation in bacteria
There are several studies available performed with structural analogue source substances investigating the potential to induce gene mutation in bacteria. All studies are taken into account by way of a Weight-of-Evidence approach.
The potential to induce gene mutation in bacteria has been investigated using Dipentaerythritol hexaesters with fatty acids, C5 and C9iso (CAS No. 647028-25-9). The study was conducted according to OECD Guideline 471 and under GLP conditions (WoE, RA-A, 647028-25-9, 1999f). The bacterial strains S. typhimurium TA 1535, TA 1537, TA 98, and TA 100 and E. coli WP2 uvr A were exposed to solutions of the test item in acetone in a plate incorporation test design. Concentrations applied in the first and second experiment were 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation. Metabolic activation was achieved by means of a cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers treated with Aroclor 1254. An oily precipitate was observed at 5000 µg/plate. Vehicle and appropriate positive controls were included into the study design. All 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 metabolising system. Investigation of the number of revertant colonies after exposure to the test item revealed no increase either in the presence nor in the absence of metabolic activation and hence the test substance was established not to induce gene mutations in the bacterial strains tested under the experimental conditions applied.
Genetic toxicity in bacteria was analysed in a GLP study performed according to OECD guideline 471 with Hexanoic acid, 2-ethyl-, 2,2-bis [ [(2-ethyl-1-oxohexyl)oxy] methyl] -1,3-propanediyl ester (CAS No. 7299-99-2) (MHWL, 2005c). Bacteria strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA were treated with concentrations of 78.1, 156, 313, 625, 1250, 2500 μg/plate (main study; precipitation at ≥ 1250 µg/plate) with and without metabolic activation by a cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone, respectively. As no increase in revertants was noted in all strains at all concentrations, the test substance was considered not mutagenic under the conditions applied. Precipitation was observed at concentrations of ≥ 1250 µg/plate at the end of the exposure period.
The mutagenic potential of Fatty acids, C5-9, tetraesters with pentaerythritol (CAS No. 67762-53-2) was tested in a reverse mutation assay according to OECD Guideline 471 and under GLP conditions (Exxon, 1999f). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA were used. Tester strains were incubated with test material dissolved in ethanol at concentrations of 33.3, 100, 333, 1000, 3330 and 5000 µg/plate with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9 mix). Vehicle and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. Thus, Fatty acids, C5-9, tetraesters with pentaerythritol did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.
An Ames test was performed with Fatty acids, C5-9, hexaesters with dipentaerythritol (CAS No. 67762-52-1) according to OECD Guideline 471 and GLP with the test substance dissolved in acetone in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and with E. coli WP2uvr A (Exxon, 1999g). Test substance concentrations of 33.3, 100, 333, 1000, 3330, and 5000 µg/plate with and without metabolic activation. Cytotoxic effects were not observed in the absence and presence of a metabolic activator. No increase in the frequency of revertant colonies compared to concurrent negative controls were observed in all tested strains, neither in the presence nor in the absence of metabolic activation. Thus, the test substance did not induce gene mutations in all tested strains under the given test conditions.
In vitro cytogenicity / chromosome aberration in mammalian cells
A relevant key study investigating the potential to induce chromosome aberration in mammalian cells performed with a suitable structural analogue source substance is available.
An in vitro mammalian chromosome aberration test was performed with Dipentaerythritol hexaesters with fatty acids, C5 and C9iso (CAS No. 647028-25-9) in cultured peripheral human lymphocytes according to OECD Guideline 473 and under GLP conditions (WoE, RA-A, 647028-25-9, 2000b). Duplicate cultures of peripheral human lymphocytes cells were evaluated for chromosome aberrations in the presence as well as the absence of metabolic activation. Metabolic activation was achieved by a cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of rats treated with Aroclor 1254. In the first experiment, cells were exposed to the test substance for 4 h followed by 20 h expression time with and without metabolic activation. The test substance was dissolved in acetone and used at concentrations of 39.06; 78.13; 156.25; 312.5; 625; 1250; 2500; 5000 µg/mL. In the second experiment cells were exposed for 20 h (without metabolic activation) and for 4 h (with metabolic activation) followed by 20 h expression time. Concentrations applied without metabolic activation were 156.25; 312.5; 625; 1250; 2500 and 5000 µg/mL and those in the experiment with metabolic activation 156.25; 312.5; 625; 1250; 2500 and 5000 µg/mL. A cloudy appearance of the test material was noted at all dose levels in both treatment groups after 4 h of exposure. In experiment 1 and 2 no dose-related toxicity was observed, a 50% mitotic inhibition was not achieved and the precipitate of the test item had no effect on the toxicity response curve. Thus, 1250, 2500 and 5000 µg/mL dose levels were selected for chromosome analysis. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. Cyclophosphamide was used as positive control material inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolising system. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations did not reveal an increase in the frequency of chromosome aberrations and polyploid cells at any dose level tested in comparison to the negative controls. The test material was therefore considered to be non-clastogenic to cultured peripheral human lymphocytes cells in vitro under the conditions of this test.
In an in vitro chromosome aberration assay, Hexanoic acid, 2-ethyl-, 2,2-bis [ [(2-ethyl-1-oxohexyl)oxy] methyl] -1,3-propanediyl ester (CAS No. 7299-99-2) was tested in Chinese Hamster Lung cells in accordance to OECD guideline 473 (MHWL, 2005d). Concentrations of 1250, 2500, 5000 μg/mL were applied to the cultured cells (main test) with and without metabolic activation by a rat liver S9-mix. Both in the absence and presence of S9-mix the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments. No cytotoxicity was observed, whereas precipitation was found at concentrations including and higher than 625 µg/mL. Thus, the target substance did not disturb mitotic processes and cell cycle progression and did not induce numerical chromosome aberrations under the experimental conditions described.
In vitro gene mutation in mammalian cells
There is one relevant key study investigating the potential to induce gene mutation in mammalian cells available. The study has been performed with a suitable structural analogue source substance.
An in vitro mammalian cell gene mutation assay according to OECD Guideline 476 and GLP was performed with Pentaerythritol tetravalerate (CAS No. 15834-04-5) in mouse lymphoma L5178Y cells (Key, RA-A, 15834-04-5, 2010). In the first experiment, the cells were treated for 3 h with 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL in the presence or absence of S9-mix (8% (v/v)). In the second experiment, concentrations of 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL were applied with metabolic activation (12%, v/v) for 3 h and 0.1, 1, 3, 10, 33, 100, 200, 250 µg/mL without metabolic activation for 24 h. The test substance was tested up to precipitating concentration (100 µg/mL and above). Cyclophosphamide and methylmethanesulfonate were used as positive controls with and without S9 mix, respectively. No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. Positive and negative controls were valid and in range of historical control data. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. It was concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.
In vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
There is one relevant study with a structural analogue source substance available investigating the potential to induce micronuclei in erythrocytes in vivo.
Fatty acids, C5-10, esters with pentraerythritol (CAS No. 68424-31-7) was found to be not genotoxic in the micronucleus assay in vivo after intraperitoneal application (Key, RA-A, 68424-31-7 1992). A single intraperitoneal injection was given to groups of 5 male and 5 female mice at a dose level of 5000 mg/kg bw. Bone marrow samples were taken 24 and 48 hours after dosing. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes over the vehicle control values were seen in either sex at either of the sampling times. Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the female animals treated with the vehicle control or with the test material. A small, but significant decrease was, however, noted in male mice treated with the test material at 5000 mg/kg bw. This small decrease is considered not to be biologically significant compared to the concurrent control values. The positive control induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.
Conclusion on genetic toxicity
Several reliable studies performed with analogue source substances are available investigating the genotoxic potential. Genotoxic effects considered include gene mutation in bacteria and mammalian cells as well as clastogenicity both in vitro and in vivo. The available data demonstrate the lack of genotoxic effects since all tests performed were negative. Thus, no hazard regarding genotoxicity is identified for the target substance Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids.
Justification for classification or non-classification
According to Article 13 of Regulation (EC) No. 1907/2006 information on intrinsic properties of substances may be generated by means other than tests, e.g. using information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI 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 read-across concept is applied to the target substance Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids from representative structural analogue source substances to avoid unnecessary animal testing.
The read-across concept is also used to derive the classification of the target substance taking the properties of the source substances into account. Based on the read-across concept, all available data on genotoxicity both in vitro and in vivo do not meet the classification criteria according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
