Registration Dossier
Registration Dossier
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EC number: 947-748-2 | 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 Fatty acids, C5-9 tetraesters with pentaerythritol (CAS No. 67762-53-2), Decanoic acid, mixed esters with octanoic acid and pentaerythritol (CAS No. 68441-68-9), Fatty acids, C16-18 (even numbered), esters with pentaerythritol (CAS No. 85116-93-4) and Fatty acids, C8-10 mixed esters with dipenaterythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8)
Chromosome aberration (OECD 473): negative in CHO cells with and without metabolic activation
Read-across from structural analogue source substance Fatty acids, C8-10 mixed esters with dipenaterythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8)
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 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:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with acceptable restrictions (shorter exposure period. Lack of data on test substance, no positive controls for 40 h time point)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted in 1997
- Deviations:
- yes
- Remarks:
- (in both experiments, cultures without metabolic activation were exposed to the test substance for about 16 h, no positive control for the 40 h time point)
- 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 containing 10% (v/v) fetal bovine serum and 2 mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Additional strain / cell type characteristics:
- other: WBL clone
- Metabolic activation:
- with and without
- Metabolic activation system:
- Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague-Dawley rats treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- 40, 80 and 160 µg/mL with and without metabolic activation
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on results of a solubility test, acetone was selected as the vehicle. The test substance was not soluble in water or dimethyl sulfoxide at any of the concentrations (10, 25, 50% (v/v)) tested. The test substance was soluble as a 50% mixture in acetone. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: - S9: N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 0.6 µg/mL (v/v) in acetone; + S9: 7,12-Dimethylbenz[a]anthracene (DMBA), 10 µg/mL (v/v) in acetone
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:
+S9: ca. 3 h (± 0.5 h)
- S9: ca. 16 h (± 0.5 h)
- Fixation time (start of exposure up to fixation or harvest of cells): ca. 16 h (± 0.5 h); second experiment - ca. 16 and 40 h (± 0.5 h)
SPINDLE INHIBITOR (cytogenetic assays): 0.2 mL Colcemid® (10 mg/mL (v/v) in cell culture medium)
STAIN (for cytogenetic assays): 5% Giemsa
NUMBER OF REPLICATIONS: 2 replications (16 h) and 1 replication (40 h), respectively
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells - Evaluation criteria:
- A test substance was considered positive in the chromosome aberration test if:
1. A statistically significant dose-related increase in the percentage of aberrant cells and in at least one of the treatment groups, the percentage of aberrant cells exceeds 5%. OR
2. A reproducible and statistically significant response for at least one of the treatment groups is observed. In addition, the mean percentage of aberrant cells exceeds 5%.
A positive result indicates that under the test conditions the test substance induces chromosomal aberrations in cultured mammalian somatic cells.
If neither of the above conditions exist, the test substance is considered nonmutagenic or negative for inducing chromosomal aberrations in this system. - Species / strain:
- Chinese hamster Ovary (CHO)
- 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
- Water solubility: The test substance is not soluble in water, therefore it was dissolved in acetone.
- Precipitation: Final concentrations of the test substance in medium of 10, 20, 39, 78, 156, 313, 625, 1250 and 2500 µg/mL were tested by visual and microscopic methods for precipitation immediately, 30 minutes and 3 h after dosing. Traces of the test substance were observed microscopically at all test concentrations equal to or greater than 78 µg/mL. Therefore, the upper limit of the culture medium solubility of the test substance was considered to be between 39 and 78 µg/mL. Based on these results, the study director selected the following concentrations for the toxicity pretest: 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL.
In the main experiments, slight precipitation was observed in the second experiment after 16 h at 160 µg/mL without metabolic activation. Precipitation was not noted at any other harvest of a 160 µg/mL culture.
RANGE-FINDING/SCREENING STUDIES: To determine a concentration selection for the aberration assay, a toxicity pretest was conducted with concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL of the test substance with and without metabolic activation. The concentrations tested were based on the results of a culture medium solubility test. The cultures with metabolic activation were treated for 3 h (± 0.5 h). The cultures without metabolic activation were treated until 2-3 h prior to harvest. All cultures were harvested about 16 h from the beginning of treatment. After harvest, the number of cells that survived treatment were counted using a hemacytometer to evaluate cytotoxicity and the mitotic indices (number of mitotic cells per 1000 total cells) were determined to evaluate cell cycle suppression. The selected concentrations for the aberration assay were based on the results of the cell count data and mitotic index data. The highest reduction in cell survival was observed at 160 µg/mL without metabolic acvtivation, where reduction in viability of 37% was noted. Other less notable reductions in cell survival were noted (see table 3), but were not indicative of a concentration-related trend. Based on these results, the concentrations selected for the aberration assay were 40, 80 and 160 µg/mL. - 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:
- 23 Aug - 1 Sep 1995
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Comparable to guideline study with acceptable restrictions (no E.coli strain or equivalent S. typhimurium strain was tested; lack of data on test substance, methodological deficiencies, no bacteria were added to the plates for tester strain TA1538 in the repeat experiment)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- (no E.coli strain or equivalent S. typhimurium strain was tested; lack of data on test substance, methodological deficiencies, no bacteria were added to the plates for tester strain TA1538 in the repeat experiment
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of Sprague-Dawley rats treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- First experiment (Initial assay): 0.5, 5, 50, 500, 5000 µg/plate with and without metabolic activation
Second experiment (Repeat assay): 50, 100, 500, 1000, 5000 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone for the test substance, DMSO for the positive control substances
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone, DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: -S9: 9-Aminoacridine (9AA; 100 µg/plate) for TA1537; N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG; 10 µg/plate) for TA100 and TA1535; 2-nitrofluorene (2NF; 5 µg/plate) for TA98 and TA1538; +S9: 2-Aminoanthracene (2AA; 2.5 µg/plate) for all strains
- Remarks:
- test substance was dissolved in acetone; positive control substances were dissolved in DMSO
- 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 with one exception: in the repeat assay, inadvertently no TA 1538 bacteria were added to the plates at 50, 100 and 500 µg/plate without S9-mix.
DETERMINATION OF CYTOTOXICITY
- Method: notable reduction of the bacterial background lawn and/or a greater than 50% reduction in the mean number of revertants in comparison to the vehicle control - Evaluation criteria:
- The test substance may be considered positive in this test system if the following criteria are met: For the test item to be considered mutagenic, three-fold (or more) increases in mean revertant numbers must be observed in comparison with vehicle control plates.
In addition, there must be evidence of a dose-related increase in the number of revertants over at least three concentrations of the test substance. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
A lack of response in the positive controls or spontaneous revertant frequencies which were not in keeping with historical laboratory values would render that portion of the test invalid. - Statistics:
- Mean values and standard deviation were calculated.
- 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:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was observed in the initial assay and the repeat assay.
- Other confounding effects: Beading of the test substance was observed in the initial assay and the repeat assay at 500 µg/plate and higher with and without metabolic activation in all strains. The only exception was the strain TA 100 in the repeat assay without metabolic activation, where a beading of the test substance was observed at 1000 and 5000 µg/plate.
RANGE-FINDING/SCREENING STUDIES: A range-finding test was conducted to determine the doses used for the main studies. Therefore, concentrations of the test substance of 0.5, 1, 5, 10, 50, 100, 500, 1000, 2000 and 5000 µg/plate were tested. Toxicity was not observed at any concentration tested. Precipitation of the test substance was observed at 1000 and 2000 µg/plate with metabolic activation and between 1 and 500 µg/plate without metabolic activation. Beading of the test substance was observed at 500 µg/plate and higher with metabolic activation and at 100 µg/plate and higher without metabolic activation. - 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:
- other: Comparable to guideline study with acceptable restrictions (no details on analytical purity given)
- 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 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
- Study period:
- 17 - 25 Apr 1991
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Guideline study with acceptable restrictions (the recommended strains TA 102 or E. coli were not included in the assay; only one positive control substance was tested for all tester strains in the presence of metabolic activation)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- the recommended strains TA 102 or E. coli were not included in the assay; only one positive control substance was tested for all tester strains in the presence of metabolic activation
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Wistar rats treated i.p. with a single dose of 500 mg/kg bw Arochlor 1254 five days prior to sacrifice
- Test concentrations with justification for top dose:
- Experiments I and II: 8, 40, 200, 1000 and 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Tween 80/bidest. water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- -S9: sodium azide (2 µg/plate, TA 100 and TA 1535); 9-aminoacridine: (80 µg/plate, TA 1537); 4-nitro-o-phenylendiamine (40 µg/plate, TA 98 and TA 1538); +S9: 2-aminoanthracene (2.5-5 µg/plate, all tester strains)
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: 4-nitro-o-phenylendiamine, 2-aminoanthracene
- 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 bacterial background lawn - Evaluation criteria:
- A combination of the following criteria was considered as a positive result:
- the plate background of non-reverted bacteria did not show any growth reduction versus the respective negative controls.
- the spontaneous mutation rates of each tester strain per plate were within the characteristic spontaneous mutation rates
- as a rule, the positive controls showed mutation rates exceeding the control values of TA 100 at least by the factor 2.0 and those of the other tester strains at least by the factor 3.0.
- at more than one dose tested, the test substance caused at least a 2.0-fold increase in comparison with the negative controls in the tester strain TA 100. For the other tester strains, an increase in the mutation rate of more than 3.0 above the corresponding negative controls was considered positive.
If the test substance induced reverse mutations in only one test, and if this effect could not be reproduced in one or several repeated tests, the initially positive test data lost their significance. - Statistics:
- Mean values and standard deviations of revertants per plate were calculated.
- 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 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:
- COMPARISON WITH HISTORICAL CONTROL DATA: the spontaneous mutation rates of each tester strain per plate were within the characteristic historical ranges, both in the presence or absence of metabolic activation
- 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:
- 16 July - 27 July 1991
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Guideline study with acceptable restrictions (Strain S. typhimurium TA102 or E.coli WP2 were not tested).
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Strain S. typhimurium TA102 or E.coli WP2 not 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
- Additional strain / cell type characteristics:
- other: rfa-, uvR-, Strains 98 and 100 also R+
- Species / strain / cell type:
- S. typhimurium TA 1538
- Additional strain / cell type characteristics:
- other: rfa-, uvR-
- 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:
- 8, 40, 200, 1000 and 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Tween 80
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- 2 µg/plate; TA100 and TA1535 (-S9)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- 80 µg/plate; TA1537 (-S9)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylendiamine
- Remarks:
- 40 µg/plate; TA98 and TA1538 (- S9)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene
- Remarks:
- all strains with metabolic activation; 2.5 µg/plate: TA1535, TA1537; 5 µg/plate: TA100, TA1538, TA98
- 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, but tested up to precipitating concentrations
- Remarks:
- Precipitate was seen at concentrations of 1000 µg/plate and higher
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results: negative
Referenceopen allclose all
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 |
Aberration frequency |
|
in µg/mL |
in % |
in % |
in % |
Exposure period 16 h, fixation time 16 h, without S9 mix |
||||
vehicle |
0.5% (v/v) |
6.8 |
0.5 |
0.5 |
MNNG |
0.6 |
6.2 |
22.5** |
24.5 |
Test substance |
40 |
5.6 |
0.5 |
0.5 |
80 |
6.5 |
0.5 |
0.5 |
|
160 |
7.2 |
1.0 |
1.0 |
|
Exposure period 3 h, fixation time 16 h, with S9 mix |
||||
Acetone |
0.5% (v/v) |
5.5 |
1.0 |
1.0 |
DMBA |
10 |
2.6 |
33.5** |
42.0 |
Test substance |
40 |
5.3 |
1.5 |
1.5 |
80 |
6.3 |
0.5 |
0.5 |
|
160 |
4.6 |
2.0 |
2.0 |
|
**statistically significantly higher than vehicle control (p<0.001)
MNNG: N-Methyl-N-Nitro-N-Nitrosoguanidine; DMBA: 7,12-Dimethylbenz[a]anthracene (positive controls)
Table 2. Test results of experiment 2
Test item |
Concentration |
Mitotic Index |
Aberrant cells |
Aberration frequency |
|
in µg/mL |
in % |
in % |
in % |
Exposure period 16 h, fixation time 16 h, without S9 mix |
||||
vehicle |
0.5% (v/v) |
7.2 |
1.0 |
1.0 |
MNNG |
0.6 |
5.4 |
17.5** |
17.5 |
Test substance |
40 |
7.1 |
1.0 |
1.0 |
80 |
5.4 |
0.5 |
0.5 |
|
160 |
7.1 |
2.5 |
2.5 |
|
Exposure period 3 h, fixation time 16 h, with S9 mix |
||||
Acetone |
0.5% (v/v) |
2.2 |
0.0 |
0.0 |
DMBA |
10 |
4.5 |
33.0** |
43.0 |
Test substance |
40 |
2.2 |
1.0 |
1.0 |
80 |
2.4 |
2.0 |
2.0 |
|
160 |
2.0 |
1.5 |
1.5 |
|
Exposure period 16 h, fixation time 40 h, without S9 mix |
||||
Acetone |
0.5% (v/v) |
3.4 |
2.5 |
2.0 |
MNNG # |
0.6 |
--- |
--- |
--- |
Test substance |
40 |
3.0 |
4.0 |
4.5 |
80 |
2.2 |
3.0 |
3.0 |
|
160 |
3.4 |
2.0 |
2.0 |
|
Exposure period 3 h, fixation time 40 h, with S9 mix |
||||
Acetone |
0.5% (v/v) |
4.8 |
2.5 |
2.5 |
DMBA # |
10 |
--- |
--- |
--- |
Test substance |
40 |
5.4 |
2.0 |
2.0 |
80 |
4.8 |
2.0 |
2.0 |
|
160 |
5.0 |
0.5 |
0.5 |
|
**statistically significantly higher than vehicle control (p<0.001)
MNNG: N-Methyl-N-Nitro-N-Nitrosoguanidine; DMBA: 7,12-Dimethylbenz[a]anthracene (positive controls)
# According to the study report, positive controls were not required for the 40 h harvest.
Table 3. Toxicity pretest results
Treatment Group |
Cell Survival in %* |
|
+ S9 |
- S9 |
|
non-treated |
107 |
102 |
vehicle |
100 |
100 |
0.625 |
122 |
114 |
1.25 |
95 |
72 |
2.5 |
121 |
88 |
5 |
108 |
68 |
10 |
111 |
108 |
20 |
89 |
102 |
40 |
99 |
93 |
80 |
78 |
93 |
160 |
120 |
63 |
* % cell survival as compared to vehicle
Table 1: Test results of the initial assay
Bacterial Reverse Mutation Assay, mean revertants colonies/plate (mutation factor) |
|||||
INITIAL ASSAY (Plate Incorporation Test) |
|||||
S9-Mix |
Without
|
||||
Test item (µg/plate) |
TA98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 1538 |
Negative control |
17.7 |
105.7 |
10.0 |
3.0 |
5.3 |
Acetone |
17.3 |
97.0 |
6.3 |
3.3 |
8.0 |
0.5 |
14.7 |
98.7 |
11.7 |
5.0 |
5.7 |
5 |
10.0 |
109.0 |
12.3 |
6.7 |
8.0 |
50 |
14.3 |
101.3 |
7.7 |
4.3 |
7.7 |
500 |
18.7 B |
122.3 B |
12.3 B |
3.3 B |
4.0 B |
5000 |
16.0 B |
114.3 B |
11.3 B |
5.3 B |
4.7 B |
DMSO |
13.0 |
112.0 |
9.3 |
4.0 |
3.3 |
9AA |
--- |
--- |
--- |
674.0* |
--- |
MNNG |
--- |
945.0* |
1089.7* |
--- |
--- |
2NF |
595.0* |
--- |
--- |
--- |
826.7* |
S9-Mix
|
With |
||||
Test item (µg/plate) |
TA98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 1538 |
Negative control |
22.3 |
111.7 |
9.3 |
4.0 |
12.3 |
Acetone |
20.3 |
119.7 |
11.7 |
6.3 |
7.0 |
0.5 |
18.3 |
126.0 |
12.3 |
4.7 |
9.3 |
5 |
17.0 |
124.3 |
7.3 |
6.0 |
13.0 |
50 |
15.7 |
129.0 |
11.0 |
5.0 |
5.3 |
500 |
25.7 B |
121.7 B |
10.7 B |
4.3 B |
9.7 B |
5000 |
18.0 B |
116.3 B |
8.7 B |
5.3 B |
14.7 B |
DMSO |
13.7 |
105.0 |
12.0 |
5.3 |
13.7 |
2AA |
338.3* |
911.3* |
113.7* |
31.7* |
471.3* |
9AA: 9-aminoacridine (100 µg/plate) MNNG: N-methyl-N-nitro-N-nitrosoguanidine (10 µg/plate) 2NF: 2-nitrofluorene (5 µg/plate) 2AA: 2 -aminoanthracene (2.5 µg/plate) |
|||||
*, p<0.05; **, p<0.01 B: test substance beaded on the plate |
|||||
Table 2: Test results of the repeat assay
Bacterial Reverse Mutation Assay, mean revertants colonies/plate (mutation factor) |
|||||
REPEAT ASSAY (Plate Incorporation Test) |
|||||
S9-Mix |
Without
|
||||
Test item (µg/plate) |
TA98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 1538 |
Negative control |
14.0 |
120.0 |
9.3 |
4.7 |
7.0 |
Acetone |
16.7 |
102.7 |
10.7 |
5.3 |
3.7 |
50 |
16.7 |
117.7 |
13.7 |
5.7 |
0# |
100 |
15.7 |
108.3 |
10.0 |
7.3 |
0# |
500 |
21.7 B |
99.0 |
12.0 B |
5.7 B |
0# |
1000 |
16.7 B |
125.3 B |
7.7 B |
4.0 B |
8.3 B |
5000 |
15.0 B |
112.3 B |
9.0 B |
3.3 B |
4.3 B |
DMSO |
16.3 |
101.7 |
10.7 |
3.7 |
6.3 |
9AA |
--- |
--- |
--- |
544.3* |
--- |
MNNG |
--- |
1644.3* |
1341.3* |
--- |
--- |
2NF |
708.3* |
--- |
--- |
--- |
1019.3* |
S9-Mix
|
With |
||||
Test item (µg/plate) |
TA98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 1538 |
Negative control |
24.0 |
108.0 |
7.3 |
6.0 |
16.0 |
Acetone |
21.0 |
123.7 |
7.0 |
4.7 |
14.7 |
50 |
25.3 |
109.3 |
12.7 |
5.0 |
10.0 |
100 |
30.3 |
110.7 |
7.0 |
6.3 |
17.3 |
500 |
27.7 B |
104.3 B |
15.3 B |
5.7 B |
16.7 B |
1000 |
24.7 B |
110.3 B |
9.7 B |
11.0 B |
19.3 B |
5000 |
22.3 B |
121.7 B |
11.3 B |
4.7 B |
13.3 B |
DMSO |
17.3 |
110.0 |
9.0 |
7.0 |
14.0 |
2AA |
270.3* |
738.3* |
70.0* |
36.7* |
241.3* |
9AA: 9 -aminoacridine (100 µg/plate) MNNG: N-methyl-N-nitro-N-nitrosoguanidine (10 µg/plate) 2NF: 2-nitrofluorene (5 µg/plate) 2AA: 2 -aminoanthracene (2.5 µg/plate) |
|||||
*, p<0.05; **, p<0.01 B: test substance beaded on the plate #: bacteria were inadvertently not added |
|||||
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 |
|||||
Table 1. Test results of experiment I (plate incorporation)
Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD) |
|||||
EXPERIMENT I |
|||||
S9-Mix |
Without
|
||||
Concentration (per plate) |
TA 1535 |
TA 100 |
TA 1537 |
TA 1538 |
TA 98 |
NC |
7 ± 2 |
68 ± 8 |
11 ± 5 |
8 ± 1 |
26 ± 8 |
SC |
10 ± 5 |
68 ± 5 |
11 ± 1 |
9 ± 3 |
29 ± 8 |
Test material |
|
||||
5000 µg |
9 ± 3 |
65 ± 9 |
11 ± 5 |
7 ± 1 |
29 ± 6 |
1000 µg |
9 ± 4 |
67 ± 11 |
13 ± 2 |
9 ± 2 |
28 ± 3 |
200 µg |
8 ± 2 |
60 ± 3 |
12 ± 2 |
6 ± 1 |
29 ± 8 |
40 µg |
9 ± 3 |
68 ± 7 |
11 ± 2 |
8 ± 0 |
31 ± 7 |
8 µg |
7 ± 1 |
66 ± 9 |
11 ± 2 |
9 ± 3 |
32 ± 2 |
PC |
|
||||
SA |
420 ± 22 |
226 ± 31 |
- |
- |
- |
9-AA |
- |
- |
817 ± 114 |
- |
- |
4-NPD |
- |
- |
- |
1668 ± 154 |
624 ± 151 |
2-AA |
9 ± 5 |
65 ± 6 |
14 ± 1 |
12 ± 1 |
29 ± 9 |
S9-Mix
|
With |
||||
Concentration (per plate) |
TA 1535 |
TA 100 |
TA 1537 |
TA 1538 |
TA 98 |
NC |
10 ± 3 |
76 ± 10 |
12 ± 2 |
17 ± 6 |
40 ± 5 |
SC |
11 ± 1 |
73 ± 11 |
8 ± 3 |
24 ± 2 |
46 ± 4 |
Test material |
|
||||
5000 µg |
12 ± 2 |
84 ± 6 |
11 ± 3 |
23 ± 2 |
49 ± 5 |
1000 µg |
13 ± 4 |
69 ± 9 |
7 ± 2 |
26 ± 3 |
41 ± 8 |
200 µg |
15 ± 4 |
83 ± 5 |
10 ± 3 |
21 ± 3 |
35 ± 7 |
40 µg |
18 ± 6 |
77 ± 2 |
9 ± 3 |
23 ± 1 |
33 ± 5 |
8 µg |
18 ± 6 |
77 ± 7 |
12 ± 1 |
18 ± 2 |
36 ± 7 |
PC |
|
|
|
|
|
SA |
88 ± 9 |
120 ± 3 |
- |
- |
- |
9-AA |
- |
- |
529 ± 117 |
- |
- |
4-NPD |
- |
- |
- |
1159 ± 81 |
621 ± 55 |
2-AA |
308 ± 9 |
1064 ± 39 |
192 ± 26 |
1561 ± 80 |
1174 ± 110 |
NC = Negative control; SC = Solvent control; PC = Positive control substances; SD = standard deviation; SA: sodium azide; 9-AA: 9-aminoacridine; 4-NPD: 4-nitro-o-phenylendiamine; 2-AA: 2-aminoanthracene |
|||||
Table 2. Test results of experiment II (plate incorporation)
Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD) |
|||||
EXPERIMENT I |
|||||
S9-Mix |
Without
|
||||
Concentration (per plate) |
TA 1535 |
TA 100 |
TA 1537 |
TA 1538 |
TA 98 |
NC |
7 ± 2 |
74 ± 6 |
10 ± 1 |
10 ± 5 |
31 ± 2 |
SC |
9 ± 3 |
69 ± 9 |
10 ± 2 |
10 ± 3 |
27 ± 3 |
Test material |
|
||||
5000 µg |
7 ± 4 |
85 ± 7 |
9 ± 4 |
9 ± 3 |
27 ± 8 |
1000 µg |
6 ± 2 |
85 ± 5 |
7 ± 2 |
12 ± 5 |
21 ± 5 |
200 µg |
12 ± 3 |
86 ± 11 |
6 ± 3 |
10 ± 4 |
31 ± 9 |
40 µg |
7 ± 4 |
76 ± 10 |
7 ± 2 |
9 ± 2 |
31 ± 5 |
8 µg |
10 ± 1 |
69 ± 3 |
8 ± 1 |
11 ± 2 |
29 ± 4 |
PC |
|
||||
SA |
342 ± 22 |
282 ± 18 |
- |
- |
- |
9-AA |
- |
- |
822 ± 117 |
- |
- |
4-NPD |
- |
- |
- |
1648 ± 98 |
693 ± 108 |
2-AA |
5 ± 2 |
85 ± 18 |
9 ± 2 |
13 ± 4 |
30 ± 5 |
S9-Mix
|
With |
||||
Concentration (per plate) |
TA 1535 |
TA 100 |
TA 1537 |
TA 1538 |
TA 98 |
NC |
10 ± 3 |
86 ± 12 |
8 ± 2 |
17 ± 3 |
27 ± 4 |
SC |
12 ± 1 |
82 ± 7 |
13 ± 2 |
18 ± 3 |
34 ± 6 |
Test material |
|
||||
5000 µg |
11 ± 4 |
82 ± 2 |
10 ± 1 |
19 ± 6 |
43 ± 8 |
1000 µg |
10 ± 5 |
85 ± 16 |
9 ± 2 |
20 ± 3 |
36 ± 9 |
200 µg |
11 ± 4 |
83 ± 5 |
11 ± 2 |
15 ± 2 |
42 ± 2 |
40 µg |
10 ± 2 |
71 ± 7 |
7 ± 1 |
13 ± 4 |
34 ± 9 |
8 µg |
6 ± 1 |
72 ± 7 |
9 ± 3 |
13 ± 2 |
36 ± 6 |
PC |
|
|
|
|
|
SA |
101 ± 6 |
119 ± 8 |
- |
- |
- |
9-AA |
- |
- |
484 ± 32 |
- |
- |
4-NPD |
- |
- |
- |
1268 ± 79 |
759 ± 22 |
2-AA |
158 ± 40 |
814 ± 88 |
134 ± 15 |
1365 ± 15 |
876 ± 81 |
NC = Negative control; SC = Solvent control; PC = Positive control substances; SD = standard deviation; SA: sodium azide; 9-AA: 9-aminoacridine; 4-NPD: 4-nitro-o-phenylendiamine; 2-AA: 2-aminoanthracene |
|||||
Table 1: Mutagenicity of the test item on bacteria - experiment I
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 |
||
- |
Buffer |
116 |
9 |
10 |
22 |
7 |
|
Vehicle |
112 |
11 |
12 |
34 |
10 |
- |
8 |
114 |
10 |
7 |
29 |
10 |
- |
40 |
111 |
10 |
8 |
34 |
7 |
- |
200 |
114 |
8 |
9 |
29 |
8 |
- |
1000 |
120 |
12 |
7 |
25 |
6 |
- |
5000 |
132 |
6 |
9 |
29 |
6 |
Positive controls - S9 |
Name |
SA |
SA |
4NP |
4NP |
9AA |
Concentrations (μg/plate) |
2.0 |
2.0 |
40 |
40 |
80 |
|
Number of colonies/plate |
818 |
621 |
1792 |
1571 |
1025 |
|
+ |
Buffer |
116 |
11 |
15 |
38 |
6 |
+ |
Vehicle |
103 |
12 |
15 |
35 |
10 |
+ |
8 |
115 |
12 |
16 |
34 |
8 |
+ |
40 |
120 |
9 |
11 |
39 |
8 |
+ |
200 |
113 |
12 |
13 |
38 |
10 |
+ |
1000 |
126 |
11 |
14 |
39 |
7 |
+ |
5000 |
130 |
13 |
12 |
3934 |
7 |
Positive controls + S9 |
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
Concentrations (μg/plate) |
5.0 |
2.5 |
5.0 |
5.0 |
2.5 |
|
Number of colonies/plate |
1594 |
210 |
1769 |
1572 |
66 |
|
4NP= 4-nitro-o-phenylendiamine
SA = sodium azide
9AA = 9 -aminoacridine
2AA = 2 -aminoanthracene
Table 2: Mutagenicity of the test item on bacteria - experiment II
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 |
||
- |
Buffer |
122 |
11 |
13 |
34 |
6 |
|
Vehicle |
127 |
13 |
9 |
39 |
13 |
- |
8 |
116 |
12 |
11 |
30 |
10 |
- |
40 |
110 |
14 |
12 |
31 |
8 |
- |
200 |
121 |
13 |
14 |
39 |
5 |
- |
1000 |
111 |
17 |
18 |
29 |
7 |
- |
5000 |
122 |
16 |
13 |
32 |
10 |
Positive controls - S9 |
Name |
SA |
SA |
4NP |
4NP |
9AA |
Concentrations (μg/plate) |
2.0 |
2.0 |
40 |
40 |
80 |
|
Number of colonies/plate |
1018 |
824 |
1929 |
1643 |
1017 |
|
+ |
Buffer |
127 |
15 |
19 |
40 |
9 |
+ |
Vehicle |
116 |
21 |
20 |
43 |
10 |
+ |
8 |
128 |
11 |
19 |
46 |
10 |
+ |
40 |
126 |
17 |
19 |
49 |
6 |
+ |
200 |
115 |
13 |
19 |
45 |
9 |
+ |
1000 |
121 |
19 |
16 |
49 |
10 |
+ |
5000 |
127 |
21 |
14 |
40 |
7 |
Positive controls + S9 |
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
Concentrations (μg/plate) |
5.0 |
2.5 |
5.0 |
5.0 |
2.5 |
|
Number of colonies/plate |
1535 |
327 |
1384 |
1515 |
64 |
|
4NP= 4-nitro-o-phenylendiamine
SA = sodium azide
9AA = 9 -aminoacridine
2AA = 2 -aminoanthracene
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 substances Pentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS No. 68424-31-7) and Fatty acids, C8-10 mixed esters with dipenaterythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8)
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Guideline study with acceptable restrictions (lack of data on test substance)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- (lack of data on test substance)
- 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 Laboratories, St. Constant, Quebec, Canada
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: 32.8 - 40.1 g (males); 27.1 - 31.8 g (females)
- Assigned to test groups randomly: yes, by a computer-generated body weight sorting program
- Housing: the animals were housed individually in suspended stainless steel and wire mesh cages with absorbent paper below the cages.
- Diet: Certified Rodent Diet # 5002 (pellets) (PMI Feeds Inc.), ad libitum
- Water: tap water, ad libitum
- Acclimation period: approximately 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES:
From: 18 Sep 1995
To: 21 Sep 1995 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle/solvent used: peanut oil
- Justification for choice of solvent/vehicle: The test substance was soluble in peanut oil at the concentrations required for this study.
- Amount of vehicle: ≤ 1 mL/100 g bw - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: The test substance was thoroughly mixed with the vehicle.
- Duration of treatment / exposure:
- three applications
- Frequency of treatment:
- approximately every 24 h
- Post exposure period:
- 24 h after the last treatment
- Dose / conc.:
- 500 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Route of administration: oral by gavage
- Dose: 20 mg/kg bw - Tissues and cell types examined:
- Tissue: bone marrow of the femur
Cell type: bone marrow cells - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: range finding study performed to find the maximum tolerated dose
DETAILS OF SLIDE PREPARATION: 2 slides per animal were prepared. The slides were stained with acridine orange and wet mounted.
METHOD OF ANALYSIS: 2000 polychromatic erythrocytes (PCEs) from each animal were examined for the presence of micronuclei. - Evaluation criteria:
- The test substance may be considered positive in this test system if at least one of the following criteria is met:
- The mean number of the micronucleated PCEs of at least one dose point is statistically different from the mean number of the micronucleated PCEs of the vehicle control. This value also must be outside the normal range of the micronucleated PCEs of the vehicle control. Additionally, a dose related statistical increase in the mean number of micronucleated PCEs must be observed.
- The mean number of the micronucleated PCEs of at least two dose points is statistically different from the mean number of the micronucleated PCEs of the vehicle control. This values also must be outside the normal range of the micronucleated PCEs of the vehicle control. - Statistics:
- The statistical analysis included means and standard deviations of the micronuclei data and a test of equality of group means performed by a standard one-way analysis of variance (ANOVA).
Residuals from the ANOVA were analyzed for normality by either Wilk's Criterion or the Kolomogorov-Smirnov Statistic. If the residuals were not normally distributed (at 0.01 level of significance) in more than 25% of the analyses then nonparametric analyses were performed. The nonparametric analyses included the Kruskal-Wallis one-way analysis of variance followed by Dunn's Summed Rank Test if differences were indicated. Dose response was evaluated by Jonkheere's test of Ordered Response. - 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:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 - 2000 mg/kg bw
- Clinical signs of toxicity in test animals: No toxicity was observed up to the highest dose tested.
- Harvest times: 24 h
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: Treatment of the animals with the test substance did not lead to a dose-dependent and statistically significant increase in micronuclei formation.
- Cytotoxicity: No cytotoxicity was observed, since there were no statistically significant decreases in the percentage of PCEs of the treated animals in comparison with the vehicle control. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- 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
Referenceopen allclose all
Table 1: Results of the in-vivo micronucleus assay in male animals
|
%PCE at sampling time |
Total micronuclei per 1000 PCEs at sampling time |
||
Exp group |
Number of animals |
Dose [mg/kg] |
24 h |
24 h |
Vehicle control (peanut oil) |
5 |
0 |
54.14 ± 6.00 |
0.6 ± 0.7 |
Positive control (cyclophosphamide) |
5 |
20 |
40.24 ± 11.18** |
18.7 ± 7.5** |
Test substance |
5 |
500 |
54.76 ± 4.81 |
0.9 ± 0.4 |
Test substance |
5 |
1000 |
51.80 ± 7.46 |
1.2 ± 0.8 |
Test substance |
5 |
2000 |
51.84 ± 8.85 |
0.8 ± 0.6 |
**statistically significant (p<0.01)
Table 2: Results of the in-vivo micronucleus assay in female animals
|
%PCE at sampling time |
Total micronuclei per 1000 PCEs at sampling time |
||
Exp group |
Number of animals |
Dose [mg/kg] |
24 h |
24 h |
Vehicle control (peanut oil) |
5 |
0 |
55.30 ± 4.72 |
1.5 ± 0.4 |
Positive control (cyclophosphamide) |
5 |
20 |
44.54 ± 5.17** |
10.5 ± 3.4** |
Test substance |
5 |
500 |
56.26 ± 5.89 |
0.6 ± 0.4* |
Test substance |
5 |
1000 |
60.68 ± 3.63 |
0.8 ± 0.7* |
Test substance |
5 |
2000 |
59.90 ± 2.7 |
0.4 ± 0.4* |
*statistically significant (p<0.05);**statistically significant (p<0.01)
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 analogue approach comprises aliphatic esters of poly-functional alcohols containing four to six reactive hydroxyl groups and one to six fatty acid chains. The read-across analogue approach contains mono constituent, multi-constituent and UVCB substances with fatty acid carbon chain lengths ranging from C5 - C20, which are mainly saturated but also mono unsaturated C16 and C18, polyunsaturated C18 and branched C5, C7 and C9, building mono-, di-, tri-, and tetra esters with the polyol. The available data allow for an accurate hazard and risk assessment of the target substance and the source substances 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 read-across analogue approach by interpolation to the target substances in accordance with Annex XI, Item 1.5, of Regulation (EC) No. 1907/2006 (REACH). In particular, for each specific endpoint the source substance or substances 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 substance and target substances are the basis of read-across.
A detailed justification for the read-across applied is provided in the technical dossier (see IUCLID Section 7.1 and 13) and within Chapter 5.1 of the CSR.
In vitro gene mutation in bacteria
There are several reliable studies available for structural analogue source substances investigating the potential to induce gene mutations in bacteria following the Ames test design.
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, 1999e). 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 in 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, the test substance did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.
Decanoic acid, mixed esters with octanoic acid and pentaerythritol (CAS No. 68441-68-9) has been assessed for its mutagenic potential in a reverse mutation assay performed according to OECD Guideline 471 and under GLP conditions (BASF, 1991d). The following Salmonella typhimurium strains were tested: TA 1535, TA 1537, TA 1538, TA 98 and TA 100. Tested strains were incubated with the test material dissolved in Tween 80/bidest. water at concentrations of 8, 40, 200, 1000 and 5000 µg/plate in the first and second experiment, with and without the addition of a metabolic activation system (Arochlor 1254 induced rat liver S9 mix). Vehicle, negative and appropriate positive controls were included in the study. The positive control substances 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. No cytotoxicity was observed. In conclusion, the test substance did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.
The mutagenic potential of Fatty acids, C16-18 (even numbered), esters with pentaerythritol (CAS No. 85116-93-4) was tested in a reverse mutation assay comparable to OECD Guideline 471 and under GLP conditions (BASF, 1991e). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 were used. Tester strains were incubated with test material dissolved in Tween 80 at concentrations of 8, 40, 200, 1000 and 5000 µg/plate (no toxicity but tested up to precipitating concentrations) with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9 mix). 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. Thus, the test substance did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.
The mutagenic potential of Fatty acids C8-10, mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8) was tested in a reverse mutation assay comparable to OECD Guideline 471 and under GLP conditions (Exxon, 1995e). The following Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 were used. Tester strains were incubated with the test material dissolved in acetone at concentrations of 0.5, 5, 50, 500, 5000 µg/plate in the first experiment and 50, 100, 500, 1000 and 5000 µg/plate in the repeat experiment with and without the addition of a metabolic activation system (Arochlor 1254 induced rat liver S9 mix). 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 negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. No cytotoxicity was observed but beading of the test substance occurred in the initial assay and repeat assay at 500 µg/plate and above with and without metabolic activation in all strains. Thus, the test substance did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.
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 Fatty acids C8-10, mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8) in Chinese hamster ovary cells (CHO cells) comparable to OECD Guideline 473 and under GLP conditions (Exxon, 1995f). Duplicate cultures of CHO cells were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). In the first experiment, cells were exposed to the test substance for 3 h and for 16 h followed by 16 h expression time with and without metabolic activation, respectively. The test substance was dissolved in acetone and used at concentrations of 40, 80 and 160 µg/mL. In the second experiment cells were again exposed for 3 h and for 16 h followed by 16 h expression time with and without metabolic activation, respectively. Additionally, cells were exposed for 3 and 16 h followed by 40 h expression time with and without metabolic activation, respectively. The same substance concentrations as in first experiment were used. The test substance did not induce cytotoxicity but a precipitate was visible in the second experiment at 160 µg/mL after 16 h incubation without metabolic activation. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. N-Methyl-N-Nitro-N-Nitrosoguanidine and 7,12-Dimethylbenz[a]anthracene were used as positive control materials inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolizing system. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level tested in comparison to the negative controls. The test material was therefore considered to be non-clastogenic to CHO cells in vitro.
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
Two relevant studies with structural analogue source substances and investigating the potential to induce micronuclei in erythrocytes in vivo are available.
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 (WoE, 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.
Fatty acids, C8-10 mixed esters with dipenaterythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8) was tested for its clastogenic potential in an in vivo micronucleus assay (Exxon, 1995g). Male and female CD-1 mice (5 per sex and dose) were administered 500, 1000, and 2000 mg/kg bw by oral gavage for three times. The period between the oral applications was approx. 24 h. Cyclophosphamide (20 mg/kg bw) as positive control substance and concurrent vehicle controls were also part of the study design. No toxicity was observed up to the highest dose tested. Bone marrow samples were taken 24 h after the last application of the test substance. No cytotoxicity was observed, since there were no statistically significant decreases in the percentage of polychromatic erythrocytes of the treated animals in comparison with the vehicle control. Treatment of the animals with the test substance did not lead to a dose-dependent and statistically significant increase in micronuclei formation. The sensitivity of the test system and the validity of the assay were demonstrated by a statistically significant increase in micronucleated polychromatic erythrocytes induced by the positive control, when compared to the vehicle control. In summary, the test substance was found not to be genotoxic in the in vivo micronucleus assay.
Conclusion on genetic toxicity
Several reliable studies performed with analogue source substances are available investigating their 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 Fatty acids lanolin, di-, tri- and tetraesters with pentaerythritol and rape fatty acid.
Justification for classification or non-classification
According to Article 13 of Regulation (EC) No. 1907/2006 (REACH) 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 physico-chemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the read-across concept is applied to the target substance Fatty acids lanolin, di-, tri- and tetraesters with pentaerythritol and rape fatty acid, data gaps can be filled by interpolation 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.
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