Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 228-067-3 | CAS number: 6107-56-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The results of the standard set of three in vitro experimental studies (bacterial mutagenicity, mammalian cell chromosome aberration, and mammalian cell gene mutation) conducted with the substance and with the valid read-across substances dilithium adipate, di-isonyl adipate, and docosonic acid, provided no evidence of any intrinsic in vitro genotoxic potential for calcium dioctanoate.
The in vitro mutagenicity of dilithium adipate was assessed in a GLP-compliant Bacterial Reverse Mutation Test, following OECD guideline 471 (WIL 2015). S. typhimurium and E. coli strains were treated with suspensions of dilithium adipate using both the Ames plate incorporation and pre-incubation methods at five dose levels in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The vehicle and positive controls confirmed the sensitivity of the assay and the efficacy of the S9 -mix.
Mammalian cells (CHL) were exposed to docosonic acid in vitro in both the absence and presence of a metabolic activation system (S9). The exposure groups included a range of dose levels that caused dose-related cytotoxic effects that were sufficient to validate the assay methodology. There was no evidnce of clastogenicity in CHL after exposure to docosonic acid and the substance was concluded to be not genotoxic in this assay.
Calcium dioctanoate was tested in a mammalian cell gene mutation assay according to OECD 476 test guideline. It was concluded that the test material did not show biologically relevant increases in mutagenic activity at the hprt locus in mouse lymphoma L5178Y cells in the absence and presence of S-9 when tested up to the maximum achievable concentration of 150 μg/mL (limited by solubility in the primary vehicle, purified water and allowing for 10% v/v additions of the formulated test article to the test system), under the experimental conditions described.In a study on a valid read-across substance,Di-isonyl adipate (DINA), the substance was tested in a mammalian cell gene mutation assay comparable to the OECD 490 test guideline. Tthe cells were exposed to DINA in both absence and presence of an induced liver enzyme metabolism system (S9). The dose levels used in all exposure conditions achieved an acceptable range of toxicity and in particular the upper dose levels induced the degree of toxicity normally accepted to be the maximum recommended to be used in this assay. No significant increase in the frequency of mutant colonies was oberseved in any exposure group, either with or without co-exposure with S9. DINA was concluded to be non-mutagenic to L5178Y mouse lymphoma cells.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal 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:
- Chinese hamster lung (CHL/IU)
- Details on mammalian cell type (if applicable):
- MEDIA USED
- Type and composition of media: Eagle-MEM liquid medium
-Properly maintained: yes - Additional strain / cell type characteristics:
- other: Not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- - source of S9
: S9 from rat liver
- method of preparation of S9 mix: S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone - Test concentrations with justification for top dose:
- -S9 mix (short-term exposure): 0, 875, 1750, 3500 µg/mL
+S9 mix (short-term exposure): 0, 875, 1750, 3500 µg/mL
-S9 mix (24 h continuous exposure): 0, 350, 700, 1400, 2800 µg/mL
-S9 mix (48-hour continuous exposure): 0, 288, 575, 1150, 2300 µg/mL - Vehicle / solvent:
- 1.0% carboxymethylcellulose sodium
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; preincubation;
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 3 days
- Exposure duration/duration of treatment: 6 (short-term exposure), 24, 48 h
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Giemsa
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 200
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG) - Evaluation criteria:
- The frequency of polyploid cells or cells with abnormal structure of each test group were determined according to the criteria of Ishidate.
- Key result
- Species / strain:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble in water, soluble in alcohol, ether, chloroform and acetone
- Precipitation and time of the determination: observed on the slide of continuous exposure high dose group
RANGE-FINDING/SCREENING STUDIES: see 'Any other information on material and method incl. tables'
HISTORICAL CONTROL DATA: this test was valid, since the frequency of chromosomal aberration in positive control was within background data. - Conclusions:
- The test substance did not induce structural chromosomal aberrations in the absence or presence of an exogenous metabolic activation system.
- Executive summary:
Mammalian cells (CHL) were exposed to docosonic acid in vitro in both the absence and presence of a metabolica activation system (S9). The exposure groups included a range of dose levels that caused dose-related cytotoxic effects that were sufficient to validate the assay methodology. There was no evidnce of clastogenicity in CHL after exposure to docosonic acid and the substance was concluded to be not genotoxic in this assay.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant, guideline study, available as an unpublished report.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Principles of method if other than guideline:
- Each S9 batch is characterized with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively Not the correct amount of 2-aminoanthracene was mentioned in the protocol. This writing error in the protocol had no effect on the results of the study.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- - Salmonella: +Histidine
- E.Coli: Tryptophan - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver S9-mix
- Test concentrations with justification for top dose:
- - Preliminary Toxicity Test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment one: 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment two: 52, 164, 512, 1600 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Milli Q water for test substance and DMSO for positive controls (except sodium azelate which used saline)
- Preparation: Test substance concentrations were used within 2 hours after preparation. - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2.5 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2.5 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 1 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 1 µg/plate in direct plate assay and 5 µg/plate in preincubation assay
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 15 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: Sodium azide: 5 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: ICR 191: 2.5 µg/plate and 2-nitrofluorene: 10 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-nitrofluorene: 10 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Methylmethanesulfonate: 650 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitroquinoline N-oxide: 10 µg/plate
- Remarks:
- Without S9 mix
- Details on test system and experimental conditions:
- METHODS OF APPLICATION
- Experiment 1: In agar (plate incorporation)
- Experiment 2: Pre-incubation
DURATION
- Preincubation period for bacterial strains: 30 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Not applicable
NUMBER OF REPLICATIONS: Triplicate plating.
DETERMINATION OF CYTOTOXICITY
- Method: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
To determine the toxicity, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. - Evaluation criteria:
- Acceptability of the assay
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Data evaluation and statistical procedures
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment. - Statistics:
- No formal hypothesis testing was done. Standard deviation was determined.
- 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:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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 nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Results: All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Precipitate: Precipitation of the substance on the plates was not observed at the start or at the end of the incubation period in all tester strains.
Toxicity: There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Mutagenicity: In the direct plate test and the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.
Negative controls: The negative control values were within the laboratory historical control data ranges.
Positive controls: The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1537 in the absence of S9-mix, second experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Based on the results of this study, it is concluded that dilithium adipate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. - Executive summary:
The in vitro mutagenicity of dilithium adipate was assessed in a GLP-compliant Bacterial Reverse Mutation Test, following OECD guideline 471 (WIL 2015). S. typhimurium and E. coli strains were treated with suspensions of dilithium adipate using both the Ames plate incorporation and pre-incubation methods at five dose levels in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The vehicle and positive controls confirmed the sensitivity of the assay and the efficacy of the S9 -mix.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Justification for type of information:
- 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance consists of calcium salts of fatty acids. Since the test item is an organic salt which dissociates in an aqueous solute, only the carboxylate (i.e. octanoate) has been considered therefore the chosen source substance is an adipate ester, rather than metal salts of fatty acids.
Adipate esters are known to be rapidly hydrolyzed in the body to form adipic acid and the corresponding alcohol [Fox et al, 1984; Guest et al, 1985; Takahashi et al, 1981]. Adipic acid was negative in a battery of genotoxicity assays [Ramel and Magnusson, 1979; Simmon and Eckford, 1978]. Available data suggest that medium-chain-length alcohols are not genotoxic [Dean et al, 1985; McCann et al, 1975]; however, iso-nonyl alcohol, the hydrolysis product of DINA, has not been evaluated. These data suggest that the metabolites of DINA, like the parent compound, are unlikely to be genotoxic.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See Test material sections of the source and target records for details.
3. ANALOGUE APPROACH JUSTIFICATION
In experimental tests for organic salts, the concentration of the counter-ion is usually not measured and only the organic ion (or its neutral form) is monitored. Moreover the medium used to conduct toxicological and ecotoxicological assays is buffered at a pH between 6 – 9, where there is an equilibrium between the non-ionized form (free acid) and the ionised form (carboxylate), and both are present in the test medium, no matter if the acid or a salt of it was introduced first. In fact, at this pH, most of the substance will be in its ionized form (carboxylate). Consequently, the toxicity properties of the test item are based on those of octanoic acid, which is a C8 fatty acid. The toxicological properties of fatty acids in general are therefore relevant to this substance.
Fatty acids are an endogenous part of every living cell and are absorbed, digested and transported in animals and humans. When taken up by tissues, they can either be stored as triglycerides or can be oxidised via the ß-oxidation and tricarboxylic acid pathways. The fatty acid moieties of calcium salts of fatty acids are natural constituents of the human body and essential components of a balanced human nutrition. Fatty acids, through their inclusion in REACH Annex V, are excluded from REACH registration requirements provided they are obtained from natural sources and not chemically modified, are not PBT or vPvB or give rise to an equivalent level of concern, and do not meet the criteria for classification as dangerous, except those classified only as flammable or skin or eye irritants.
Further information is included in the read-across justification attached to Section 13 of IUCLID. - Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- not applicable
- Principles of method if other than guideline:
- The mouse lymphoma gene mutation assay was conducted essentially as described by Clive and co-workers [1973, 1979; Clive and Spector, 1975]:
Clive D, Flamm WG, Patterson JG (`1973): Specific-locus mutational assay systems for mouse lymphoma cells.
Clive D, Johnson KO, Spector JFS, Batson Ag, Brown MM (1979): Validation and characterization of the L5178Y TK +/- mouse lymphoma mutagen assay system.
Clive D, Spector JFS (1975): Laboratory proceduresfor assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. - GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Specific details on test material used for the study:
- The test sample, JAYFLEX DINA (CAS number 33703-08-1) was provided by Exxon Chemical Americas, Houston, TX. Sample purity exceeded 99% as determined by gas chromatography.
- Target gene:
- Thymidine kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- L5178Y TK +/- cells
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Fisher medium supplemented with 0.1% pluronic and 10% heat-inactivated horse serum (F10P).
Trifluorothymidine (TFT) at 3 ug/ml was used as the selective agent for TK mutants. - Metabolic activation:
- with and without
- Metabolic activation system:
- Activation by 9,000 g supernatant derived from livers of Aroclor 1254-induced male Sprague-Dawley rats.
- Test concentrations with justification for top dose:
- Non-activated: 7.5, 10, 13, 18, 24, 56, 75 and 100 µl/ml
Activated: 5.6, 10, 13, 18, 24, 56, 75 and 100 µl/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Suspension cultures of mouse lymphoma cells, heterozygous for thymidine kinase activity, were grown in Fisher medium for leukemic mouse cells supplemented with 0.1% pluronic and 10% heat-inactivated horse serum (F10P) and were exposed to test substances in the same medium. Treated cells were grown in F10P for 48 hr to allow mutation expression. Approximately 3 x 10 6 cells from each culture were then plated in a selective medium containing 3 µg/ml trifluorothymidine (TFT) to select for mutant clones. Appropriately diluted cells from each culture were also seeded in plates without TFT for viability determinations (200 cells/plate). Mutant and total colony counts at each dose point were determined by triplicate plating. Colonies were counted with an automatic colony counter; differential sizing was not performed.
The validity of the experimental assay was verified through the use of concurrent positive control materials, 7,12-dimethyl-benzanthracene (DMBA) and ethylmethane sulfonate (EMS). Increased mutation frequencies induced by the positive control materials under appropriate conditions demonstrated both the integrity of the metabolic activation system and the responsiveness of the assay system as compared to its historical performance in the laboratory. - Evaluation criteria:
- A significant increase in mutant frequency was defined as >/= 2x the mean of the concurrent vehicle controls.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks:
- EMS
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks:
- DMBA
- Additional information on results:
- DINA did not exhibit any evidence of genetic activity in the mouse lymphoma assay over a range of toxic and nontoxic doses in either the presence or absence of metabolic activation. The positive control materials, EMS and DMBA, responded appropriately, confirming the validity of this assay. These results indicated that DINA was not mutagenic in mammalian cells under the conditions of this assay.
- Conclusions:
- DINA did not show evidence of mutagenic potential in the mouse lymphoma TK +/- gene mutation assay.
- Executive summary:
Di-isonyl adipate (DINA) was tested in a mammalian cell gene mutation assay comparable to the OECD 490 test guideline. The cells were exposed to DINA in both absence and presence of an induced liver enzyme metabolism system (S9). The dose levels used in all exposure conditions achieved an acceptable range of toxicity and in particular the upper dose levels induced the degree of toxicity normally accepted to be the maximum recommended to be used in this assay. No significant increase in the frequency of mutant colonies was oberseved in any exposure group, either with or without co-exposure with S9. DINA was concluded to be non-mutagenic to L5178Y mouse lymphoma cells.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- not applicable
- Principles of method if other than guideline:
- The mouse lymphoma gene mutation assay was conducted essentially as described by Clive and co-workers [1973, 1979; Clive and Spector, 1975]:
Clive D, Flamm WG, Patterson JG (`1973): Specific-locus mutational assay systems for mouse lymphoma cells.
Clive D, Johnson KO, Spector JFS, Batson Ag, Brown MM (1979): Validation and characterization of the L5178Y TK +/- mouse lymphoma mutagen assay system.
Clive D, Spector JFS (1975): Laboratory proceduresfor assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. - GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Specific details on test material used for the study:
- The test sample, JAYFLEX DINA (CAS number 33703-08-1) was provided by Exxon Chemical Americas, Houston, TX. Sample purity exceeded 99% as determined by gas chromatography.
- Target gene:
- Thymidine kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- L5178Y TK +/- cells
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Fisher medium supplemented with 0.1% pluronic and 10% heat-inactivated horse serum (F10P).
Trifluorothymidine (TFT) at 3 ug/ml was used as the selective agent for TK mutants. - Metabolic activation:
- with and without
- Metabolic activation system:
- Activation by 9,000 g supernatant derived from livers of Aroclor 1254-induced male Sprague-Dawley rats.
- Test concentrations with justification for top dose:
- Non-activated: 7.5, 10, 13, 18, 24, 56, 75 and 100 µl/ml
Activated: 5.6, 10, 13, 18, 24, 56, 75 and 100 µl/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Suspension cultures of mouse lymphoma cells, heterozygous for thymidine kinase activity, were grown in Fisher medium for leukemic mouse cells supplemented with 0.1% pluronic and 10% heat-inactivated horse serum (F10P) and were exposed to test substances in the same medium. Treated cells were grown in F10P for 48 hr to allow mutation expression. Approximately 3 x 10 6 cells from each culture were then plated in a selective medium containing 3 µg/ml trifluorothymidine (TFT) to select for mutant clones. Appropriately diluted cells from each culture were also seeded in plates without TFT for viability determinations (200 cells/plate). Mutant and total colony counts at each dose point were determined by triplicate plating. Colonies were counted with an automatic colony counter; differential sizing was not performed.
The validity of the experimental assay was verified through the use of concurrent positive control materials, 7,12-dimethyl-benzanthracene (DMBA) and ethylmethane sulfonate (EMS). Increased mutation frequencies induced by the positive control materials under appropriate conditions demonstrated both the integrity of the metabolic activation system and the responsiveness of the assay system as compared to its historical performance in the laboratory. - Evaluation criteria:
- A significant increase in mutant frequency was defined as >/= 2x the mean of the concurrent vehicle controls.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks:
- EMS
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks:
- DMBA
- Additional information on results:
- DINA did not exhibit any evidence of genetic activity in the mouse lymphoma assay over a range of toxic and nontoxic doses in either the presence or absence of metabolic activation. The positive control materials, EMS and DMBA, responded appropriately, confirming the validity of this assay. These results indicated that DINA was not mutagenic in mammalian cells under the conditions of this assay.
- Conclusions:
- DINA did not show evidence of mutagenic potential in the mouse lymphoma TK +/- gene mutation assay.
- Executive summary:
Di-isonyl adipate (DINA) was tested in a mammalian cell gene mutation assay comparable to the OECD 490 test guideline. The cells were exposed to DINA in both absence and presence of an induced liver enzyme metabolism system (S9). The dose levels used in all exposure conditions achieved an acceptable range of toxicity and in particular the upper dose levels induced the degree of toxicity normally accepted to be the maximum recommended to be used in this assay. No significant increase in the frequency of mutant colonies was oberseved in any exposure group, either with or without co-exposure with S9. DINA was concluded to be non-mutagenic to L5178Y mouse lymphoma cells.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Justification for type of information:
- 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance consists of calcium salts of fatty acids. Since the test item is an organic salt which dissociates in an aqueous solute, only the carboxylate (i.e. octanoate) has been considered therefore the chosen source substance consists of C22 fatty acids, rather than metal salts of fatty acids. The fatty acids differ by chain length, with the source substance being C22 and the target substance being C8. However, these will all break down to intermediate fatty acids, including glycerol, and finally carbon dioxide and water.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See Test material sections of the source and target records for details.
3. ANALOGUE APPROACH JUSTIFICATION
In experimental tests for organic salts, the concentration of the counter-ion is usually not measured and only the organic ion (or its neutral form) is monitored. Moreover the medium used to conduct toxicological and ecotoxicological assays is buffered at a pH between 6 – 9, where there is an equilibrium between the non-ionized form (free acid) and the ionised form (carboxylate), and both are present in the test medium, no matter if the acid or a salt of it was introduced first. In fact, at this pH, most of the substance will be in its ionized form (carboxylate). Consequently, the toxicity properties of the test item are based on those of octanoic acid, which is a C8 fatty acid. The toxicological properties of fatty acids in general are therefore relevant to this substance.
Fatty acids are an endogenous part of every living cell and are absorbed, digested and transported in animals and humans. When taken up by tissues, they can either be stored as triglycerides or can be oxidised via the ß-oxidation and tricarboxylic acid pathways. The fatty acid moieties of calcium salts of fatty acids are natural constituents of the human body and essential components of a balanced human nutrition. Fatty acids, through their inclusion in REACH Annex V, are excluded from REACH registration requirements provided they are obtained from natural sources and not chemically modified, are not PBT or vPvB or give rise to an equivalent level of concern, and do not meet the criteria for classification as dangerous, except those classified only as flammable or skin or eye irritants.
Further information is included in the read-across justification attached to Section 13 of IUCLID. - Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal 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:
- Chinese hamster lung (CHL/IU)
- Details on mammalian cell type (if applicable):
- MEDIA USED
- Type and composition of media: Eagle-MEM liquid medium
-Properly maintained: yes - Additional strain / cell type characteristics:
- other: Not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- - source of S9
: S9 from rat liver
- method of preparation of S9 mix: S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone - Test concentrations with justification for top dose:
- -S9 mix (short-term exposure): 0, 875, 1750, 3500 µg/mL
+S9 mix (short-term exposure): 0, 875, 1750, 3500 µg/mL
-S9 mix (24 h continuous exposure): 0, 350, 700, 1400, 2800 µg/mL
-S9 mix (48-hour continuous exposure): 0, 288, 575, 1150, 2300 µg/mL - Vehicle / solvent:
- 1.0% carboxymethylcellulose sodium
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; preincubation;
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 3 days
- Exposure duration/duration of treatment: 6 (short-term exposure), 24, 48 h
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Giemsa
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 200
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG) - Evaluation criteria:
- The frequency of polyploid cells or cells with abnormal structure of each test group were determined according to the criteria of Ishidate.
- Key result
- Species / strain:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble in water, soluble in alcohol, ether, chloroform and acetone
- Precipitation and time of the determination: observed on the slide of continuous exposure high dose group
RANGE-FINDING/SCREENING STUDIES: see 'Any other information on material and method incl. tables'
HISTORICAL CONTROL DATA: this test was valid, since the frequency of chromosomal aberration in positive control was within background data. - Conclusions:
- The test substance did not induce structural chromosomal aberrations in the absence or presence of an exogenous metabolic activation system.
- Executive summary:
Mammalian cells (CHL) were exposed to docosonic acid in vitro in both the absence and presence of a metabolic activation system (S9). The exposure groups included a range of dose levels that caused dose-related cytotoxic effects that were sufficient to validate the assay methodology. There was no evidnce of clastogenicity in CHL after exposure to docosonic acid and the substance was concluded to be not genotoxic in this assay.
The study is considered to be relevant and reliable for use for this endpoint.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 19 June 2020 to 29 July 2020
- 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 using the Hprt and xprt genes)
- GLP compliance:
- yes
- Remarks:
- See also notes on stability below. Stability, homogeneity and achieved concentration of test article formulations were not analysed in this study however formulations were used on the day of preparation.
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Supplied by sponsor; batch number: 20-02934-02
- Expiration date of the lot/batch: Not applicable
- Purity test date: 100 %
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored under nitrogen and protected from light
- Stability under test conditions: Stable (The stability of the Calcium dioctanoate sample, as received, could not be proven definitively. Published literature states that in aqueous solution, Calcium dioctanoate ionises to calcium ions and octanoate ions. Calcium ions are stable and so is octanoic acid (also known as caprylic acid) and a Safety Data Sheet for caprylic acid gives a 60 month shelf life. It may therefore be predicted that Calcium dioctanoate will be stable, as its counter ions.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: soluble in purified water.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Preliminary solubility data indicated that test material was poorly soluble or insoluble in dimethyl sulfoxide, dimethyl formamide, acetone, and ethanol, and the solubility when added directly to culture medium was below 0.2 mg/mL. However, the test article was soluble in purified water at a concentration of 1.5 mg/mL. A 10% v/v dilution of the formulated test article at 1.5 mg/mL into culture medium to give a final concentration of 150 µg/mL resulted in no visible precipitation, either immediately upon addition or over a period of approximately 3 hours, with warming at 37°C.
Test article stock solutions were prepared by formulating the test material under subdued lighting in purified water, with the aid of vortex mixing, warming at 37°C and ultrasonication for 15 minutes (where required), to give the maximum required concentration. The stock solutions were membrane filter-sterilised (Pall Acrodisc 32 mm, 0.2 µm pore size) and subsequent dilutions made using purified water. The test article solutions were protected from light and used within approximately 1.5 hours of initial formulation.
Formulations Analysis
In accordance with the regulatory test guidelines applicable for this study, no analyses of the stability of the test article in administered formulations or dilutions was undertaken as fresh preparation of test article were employed.
Following discussions with the Sponsor, analyses for achieved concentration and homogeneity (where appropriate) of test article formulations were not conducted as part of this study, as this is not a requirement of the regulatory test guidelines.
The absence of such analyses is noted in the Study Director’s statement of GLP compliance. - Target gene:
- HPRT gene
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells
Mouse lymphoma L5178Y systems are statistically more sensitive than CHO and V79 systems (Arlett et al., 1989)
For cell lines:
Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free.
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and placed in an incubator set to 37ºC. When the cells were growing well, subcultures were established in an appropriate number of flasks.
MEDIA USED
RPMI 1640 media supplied containing L-glutamine and HEPES and prepared as follows:
Final Concentration in:
RPMI A RPMI 10 RPMI 20
Horse serum
(heat inactivated) 0% v/v 10% v/v 20% v/v
Penicillin 100 units/mL 100 µg/mL 100 units/mL
Streptomycin 100 µg/mL 100 units/mL 100 µg/mL
Amphotericin B 2.5 µg/mL 2.5 µg/mL 2.5 µg/mL
Pyruvic Acid 0.2 mg/mL 0.2 mg/mL 0.2 mg/mL
Pluronic 0.5 mg/mL 0.5 mg/mL -
RPMI 5 consisted of RPMI 10 diluted with RPMI A [prepared as RPMI 10 but with no serum added] to give a final concentration of 5% serum. - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: Aroclor 1254 induced S9
- source of S9: Molecular Toxicology Inc.
- method of preparation of S9 mix : Buffer with NADP and salts of Mg and K.
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9 mix in final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Fully characterized by the supplier, QC statement included in the report. - Test concentrations with justification for top dose:
- Seven concentrations from 10 to 150 ug/mL, the highest dose level being limited by solubility in the primary vehicle, purified water.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Purified water
- Justification for choice of solvent/vehicle: The test item was soluble in water
- Justification for percentage of solvent in the final culture medium: 10% which is acceptable for an aqueous vehicle - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- purified water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- with metabolic activation
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- 3.00 µg/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- purified water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- with metabolic activation
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- 2.00 µg/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- purified water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- without metabolic activation
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- 0.20 µg/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- purified water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- without metabolic activation
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- 0.15 µg/mL
- Details on test system and experimental conditions:
- Cytotoxicity Range-Finder Experiment
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment volume was 20 mL.
Following 3 hour treatment, cells were centrifuged (200 g), washed with tissue culture medium and resuspended in 20 mL RPMI 10.
Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, for 8 days. Wells containing viable clones were identified by eye using background illumination and counted.
Mutation Assay
Treatment of Cell Cultures
At least 10E7 cells in a volume of 16 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 2 mL vehicle or test article or 0.2 mL of the positive control solution (plus 1.8 mL purified water) was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours in an incubator set to 37°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10 medium. Cell densities were determined using a Coulter counter and the concentrations adjusted to 2 x 10 5 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies (Brusick, 1986; Scott et al., 1991). Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.
Plating for Survival
Following adjustment of the cultures to 2 x 10 5 cells/mL after treatment, samples from these were diluted to 8 cells/mL as shown in table 2 (below in "Any other information on results incl. tables" ).
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air until scoreable (7 days). Wells containing viable clones were identified by eye using background illumination and counted.
Expression Period
Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask. From observations on recovery and growth of the cultures during the expression period, the following cultures were selected to be plated for viability and 6TG resistance as shown in the table 3 (below in "Any other information on results incl. tables" ).
Plating for Viability
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10 5 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL as shown in table 4 (below in "Any other information on results incl. tables" ).
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air until scoreable (8 days). Wells containing viable clones were identified by eye using background illumination and counted.
Plating for 6TG Resistance
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10 5 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 µg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 10 4 cells/well). Plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (12 days). Wells containing clones were identified as above and counted.
Analysis of Results: Please see "Any other information on results incl. tables" for details. - Rationale for test conditions:
- Acceptance Criteria
The assay was considered valid if all of the following criteria were met:
1. The MF in the vehicle control cultures was considered acceptable for addition to the laboratory historical negative control database
2. The MF in the concurrent positive controls induced responses that were comparable with those generated in the historical positive control database and gave a clear, unequivocal increase in MF over the concurrent negative control
3. The test was performed with and without metabolic activation
4. Adequate numbers of cells and concentrations were analysable. - Evaluation criteria:
- For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF +/- 2 standard deviations.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. - Statistics:
- Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- 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:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Toxicity
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 4.688 to 150 µg/mL (limited by solubility in the primary vehicle, purified water). No evidence of precipitation was observed either immediately upon addition of the test article or following 3 hours of incubation at 37ºC. The highest concentration tested, 150 µg/mL, gave 84% and 176% RS in the absence and presence of S-9, respectively (see table 1. below in "Any other information on results incl. tables" ). There was a possible dilution error prior to plating the highest concentration analysed in the presence of S 9, resulting in 176% RS at 150 µg/mL. However, there was no evidence of marked cytotoxicity over the remainder of the concentration range tested and 150 µg/mL was selected as the maximum concentration tested in the absence and presence of S-9 in the Mutation Experiment.
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations tested (150 µg/mL), compared to the concurrent vehicle controls (measured data not reported).
In the Mutation Experiment seven concentrations, ranging from 10 to 150 µg/mL, were tested in the absence and presence of S-9. No evidence of precipitation was observed either immediately upon addition of the test article or following 3 hours of incubation at 37ºC. Seven days after treatment all concentrations in the absence and presence of S-9 were selected to determine viability and 6TG resistance. The highest concentration analysed was 150 µg/mL, which gave 82% and 102% RS in the absence and presence of S-9, respectively (see table 1. below in "Any other information on results incl. tables" ).
Mutation
When tested up to the maximum achievable concentration of 150 µg/mL for 3 hours in the absence of S-9, a statistically significant increase in MF, compared to the vehicle control value, was observed at the highest concentration tested (150 µg/mL), but there was no statistically significant linear trend. The MF value at 150 µg/mL (4.48 mutants/10 6 viable cells) was well within the historical vehicle control range generated by the last 20 experiments (mean ± 2 standard deviations) performed in this laboratory (0.98 to 10.61 mutants/10 6 viable cells: mean value 5.80) and was consistent with published HPRT data. The statistically significant increase was attributable to the very low mean vehicle control MF in this experiment (1.09 mutants/10 6 viable cells), which was at the lower end of the historical vehicle control range. Although this observation was at the highest concentration analysed and was a non-toxic concentration (limited by solubility in the primary vehicle) under the conditions of the test it is considered to be not biologically relevant. The MF value was well within the historical vehicle control at all test article concentrations analysed and the magnitude of the increase in MF at 150 µg/mL was very small and actually less than the mean of the historical vehicle control range.
When tested up to the maximum achievable concentration of 150 µg/mL for 3 hours in the presence of S-9, statistically significant increases in MF, compared to the vehicle control value, were observed at two intermediate concentrations (40 and 80 µg/mL) and there was a statistically significant linear trend (p≤0.05). However, the statistically significant increases in MF were observed at intermediate concentrations, therefore there was no real concentration-related relationship over the range under test. The MF values at 40 and 80 µg/mL (5.68 and 6.82 mutants/10 6 viable cells) were both well within the historical vehicle control range generated by the last 20 experiments (mean ± 2 standard deviations) performed in this laboratory (0 to 12.50 mutants/10 6 viable cells: mean value 5.92) and were consistent with published HPRT data. The statistical significance of the increases observed at 40 and 80 µg/mL were attributable to the very low mean vehicle control MF (1.15 mutants/10 6 viable cells), which was at the lower end of the historical vehicle control range. Overall, the statistically significant increases in MF were observed at intermediate concentrations analysed, the MF values at both concentrations were within the current historical vehicle control range and the magnitude of these increases in MF were small. Under the conditions of the test, these observations are considered to be not biologically relevant.
6‑TG resistant mutants/10 6 viable cells 7 days after treatment- Conclusions:
- It is concluded that the test material did not show biologically relevant increases in mutagenic activity at the hprt locus in mouse lymphoma L5178Y cells in the absence and presence of S-9 when tested up to the maximum achievable concentration of 150 µg/mL (limited by solubility in the primary vehicle, purified water and allowing for 10% v/v additions of the formulated test article to the test system), under the experimental conditions described.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant, guideline study, available as an unpublished report.
- Justification for type of information:
- Source and target substances are likely to dissociate after administration into metals ions and fatty acids due to the ionic bond between the metal cation and fatty acid ion being disrupted by polar aqueous media. The fatty acid component is not expected to be hazardous as it is potentially exempt from REACH under Annex V. The main difference between source and target substances is the cation and therefore test organisms would be exposed to either calcium from the target substance or lithium from the source substance. Although the cations are different, read across from lithium adipate is considered to be appropriate for in vitro genetic toxicity and skin sensitisation as both lithium and calcium show a similar lack of toxicity for these endpoints. Further information is included in the read-across justification attached to Section 13 of IUCLID.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Principles of method if other than guideline:
- Each S9 batch is characterized with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively Not the correct amount of 2-aminoanthracene was mentioned in the protocol. This writing error in the protocol had no effect on the results of the study.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- - Salmonella: +Histidine
- E.Coli: Tryptophan - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver S9-mix
- Test concentrations with justification for top dose:
- - Preliminary Toxicity Test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment one: 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment two: 52, 164, 512, 1600 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Milli Q water for test substance and DMSO for positive controls (except sodium azelate which used saline)
- Preparation: Test substance concentrations were used within 2 hours after preparation. - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2.5 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2.5 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 1 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 1 µg/plate in direct plate assay and 5 µg/plate in preincubation assay
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 15 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: Sodium azide: 5 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: ICR 191: 2.5 µg/plate and 2-nitrofluorene: 10 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-nitrofluorene: 10 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Methylmethanesulfonate: 650 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitroquinoline N-oxide: 10 µg/plate
- Remarks:
- Without S9 mix
- Details on test system and experimental conditions:
- METHODS OF APPLICATION
- Experiment 1: In agar (plate incorporation)
- Experiment 2: Pre-incubation
DURATION
- Preincubation period for bacterial strains: 30 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Not applicable
NUMBER OF REPLICATIONS: Triplicate plating.
DETERMINATION OF CYTOTOXICITY
- Method: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
To determine the toxicity, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. - Evaluation criteria:
- Acceptability of the assay
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Data evaluation and statistical procedures
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment. - Statistics:
- No formal hypothesis testing was done. Standard deviation was determined.
- 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:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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 nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Results: All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Precipitate: Precipitation of the substance on the plates was not observed at the start or at the end of the incubation period in all tester strains.
Toxicity: There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Mutagenicity: In the direct plate test and the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.
Negative controls: The negative control values were within the laboratory historical control data ranges.
Positive controls: The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1537 in the absence of S9-mix, second experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Based on the results of this study, it is concluded that dilithium adipate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. - Executive summary:
The in vitro mutagenicity of dilithium adipate was assessed in a GLP-compliant Bacterial Reverse Mutation Test, following OECD guideline 471 (WIL 2015). S. typhimurium and E. coli strains were treated with suspensions of dilithium adipate using both the Ames plate incorporation and pre-incubation methods at five dose levels in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The vehicle and positive controls confirmed the sensitivity of the assay and the efficacy of the S9 -mix.
Referenceopen allclose all
Table 1: Results of growth inhibition test
Test item |
Concentration in µg/mL |
Survival in % |
Exposure period 24 h, without S9 mix |
||
1% CMC▪Na |
|
100 |
Test substance |
272 |
97.7 |
454 |
95.2 |
|
756 |
96.5 |
|
1260 |
83.1 |
|
2100 |
62.1 |
|
3500 |
37.8 |
|
Exposure period 48 h, without S9 mix |
||
1% CMC▪Na |
|
100 |
Test substance |
272 |
101.1 |
454 |
104.6 |
|
756 |
98.9 |
|
1260 |
89.7 |
|
2100 |
75.9 |
|
3500 |
1.3 |
|
Exposure period 6 h, without S9 mix |
||
1% CMC▪Na |
|
100 |
Test substance |
272 |
109.6 |
454 |
93.7 |
|
756 |
102.5 |
|
1260 |
110.7 |
|
2100 |
89.8 |
|
3500 |
95.1 |
|
Exposure period 6 h, with S9 mix |
||
1% CMC▪Na |
|
100 |
Test substance |
272 |
84.3 |
454 |
85.9 |
|
756 |
85.3 |
|
1260 |
86.5 |
|
2100 |
68.3 |
|
3500 |
76.3 |
|
Table 2: Results of chromosome aberration test
Test item |
Concentration |
Aberrant cells in % |
Polyploid cells in % |
||
|
in µg/mL |
with gaps |
without gaps |
||
Exposure period 24 h, without S9 mix |
|||||
1% CMC▪Na |
|
0.5 |
0.5 |
0.0 |
|
MMC |
0.05 |
56.0 |
52.5 |
0.5 |
|
Test substance |
350 |
1.0 |
1.0 |
0.5 |
|
700 |
1.0 |
0.5 |
0.5 |
||
1400 |
0.5 |
0.0 |
0.0 |
||
2800 |
Toxic |
||||
Exposure period 48 h, without S9 mix |
|||||
1% CMC▪Na |
|
0.0 |
0.0 |
0.5 |
|
MMC |
0.025 |
58.5 |
54.5 |
0.0 |
|
Test substance |
288 |
1.0 |
1.0 |
0.0 |
|
575 |
0.5 |
0.0 |
0.5 |
||
1150 |
2.0 |
1.5 |
0.0 |
||
2300 |
Toxic |
||||
Exposure period 6 h, without S9 mix |
|||||
1% CMC▪Na |
|
1.5 |
0.5 |
0.5 |
|
CP |
12.5 |
0.5 |
0.0 |
0.5 |
|
Test substance |
875 |
0.5 |
0.5 |
0.0 |
|
1750 |
3.0 |
2.5 |
0.0 |
||
3500 |
1.0 |
0.5 |
0.5 |
||
Exposure period 6 h, with S9 mix |
|||||
1% CMC▪Na |
|
1.5 |
1.0 |
0.0 |
|
CP |
12.5 |
63.5 |
61.5 |
0.0 |
|
Test substance |
875 |
2.5 |
1.5 |
1.0 |
|
1750 |
2.0 |
2.0 |
0.0 |
||
3500 |
2.0 |
2.0 |
0.0 |
||
CMC▪Na: carboxymethylcellulose sodium (solvent)
MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)
Table 1: Dose range finding test - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay
Mean number of revertant colonies/3 replicate plates (±S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain
|
Without S9 |
With S9 |
||
Dose |
TA100 |
WP2uvrA |
TA100 |
WP2uvrA |
Positive control |
862 ± 37 |
1574 ± 37 |
1395 ± 94 |
311 ± 6 |
Solvent control |
102 ± 11 |
28 ± 6 |
82 ± 11 |
41 ± 10 |
1.7 |
88 ± 6 |
33 ± 7 |
93 ± 20 |
35 ± 8 |
5.4 |
81 ± 10 |
26 ± 6 |
105 ± 7 |
36 ± 12 |
17 |
89 ± 15 |
27 ± 9 |
102 ± 11 |
33 ± 10 |
52 |
104 ± 18 |
31 ± 4 |
104 ± 9 |
38 ± 8 |
164 |
95 ± 3 |
26 ± 7 |
105 ± 10 |
37 ± 7 |
512 |
99 ± 20 |
31 ± 1 |
95 ± 8 |
42 ± 10 |
1600 |
98 ± 16 |
33 ± 13 |
103 ± 18 |
39 ± 6 |
5000 |
87 ± 14 * |
28 ± 9 * |
93 ± 0 * |
39 ± 7 * |
* No precipitate and normal bacterial background lawn
Table 2: Experiment 1 - Mutagenic responnse in Salmonella typhimurium reverse mutation assay - Direct plate assay
Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium
|
Without S9 |
With S9 |
||||
Dose |
TA 1535 |
TA 1537 |
TA 98 |
TA 1535 |
TA 1537 |
TA 98 |
Positive control |
851 ± 29 |
421 ± 62 |
949 ± 12 |
284 ± 16 |
454 ± 42 |
1121 ± 42 |
Solvent control |
20 ± 6 |
16 ± 5 |
18 ± 2 |
15 ± 5 |
5 ± 0 |
28 ± 5 |
52 |
20 ± 10 |
8 ± 3 |
16 ± 2 |
15 ± 6 |
14 ± 6 |
39 ± 3 |
164 |
23 ± 5 |
8 ± 2 |
17 ± 8 |
12 ± 2 |
11 ± 1 |
31 ± 9 |
512 |
17± 2 |
6 ± 6 |
12 ± 3 |
17 ± 5 |
10 ± 6 |
32 ± 7 |
1600 |
18 ± 2 |
9 ± 2 |
19 ± 4 |
25 ± 2 |
10 ± 5 |
29 ± 8 |
5000 |
12 ± 0 * |
11 ± 3 * |
17 ± 4 * |
18 ± 4 * |
9 ± 4 * |
30 ± 1 * |
* No precipitate and normal bacterial background lawn
Table 3: Experiment 2 - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay - Pre-incubation assay
Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
|
Without S9 |
With S9 |
||||||||
Dose |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2uvra |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2uvra |
Positive control |
766 ± 52 |
56 ± 5 |
1003 ± 56 |
682 ± 64 |
169 ± 22 |
126 ± 9 |
160 ± 8 |
432 ± 26 |
2266 ± 183 |
399 ± 21 |
Solvent control |
17 ± 3 |
10 ± 6 |
16 ± 4 |
103 ± 13 |
33 ± 3 |
8 ± 2 |
12 ± 4 |
28 ± 3 |
115 ± 17 |
35 ± 5 |
52 |
17 ± 2 |
7 ± 3 |
19 ± 2 |
119 ± 19 |
26 ± 8 |
10 ± 5 |
13 ± 5 |
29 ± 3 |
126 ± 10 |
43 ± 16 |
164 |
16 ± 4 |
8 ± 4 |
19 ± 1 |
107 ± 10 |
27 ± 7 |
10 ± 5 |
12 ± 6 |
28 ± 3 |
110 ± 7 |
35 ± 5 |
512 |
11 ± 6 |
13 ± 2 |
15 ± 7 |
103 ± 11 |
30 ± 7 |
10 ± 5 |
9 ± 6 |
23 ± 2 |
109 ± 13 |
28 ± 5 |
1600 |
15 ± 4 |
9 ± 2 |
19 ± 7 |
103 ± 7 |
23 ± 1 |
10 ± 5 |
13 ± 11 |
33 ± 9 |
103 ± 5 |
42 ± 6 |
5000 |
17 ± 2 * |
9 ± 6 * |
18 ± 5 * |
107 ± 11 * |
29 ± 6 * |
10 ± 5 * |
15 ± 5 * |
25 ± 4 * |
112 ± 15 * |
28 ± 12 * |
* No precipitate and normal bacterial background lawn
Table 1: Results of growth inhibition test
Test item |
Concentration in µg/mL |
Survival in % |
Exposure period 24 h, without S9 mix |
||
1% CMC▪Na |
|
100 |
Test substance |
272 |
97.7 |
454 |
95.2 |
|
756 |
96.5 |
|
1260 |
83.1 |
|
2100 |
62.1 |
|
3500 |
37.8 |
|
Exposure period 48 h, without S9 mix |
||
1% CMC▪Na |
|
100 |
Test substance |
272 |
101.1 |
454 |
104.6 |
|
756 |
98.9 |
|
1260 |
89.7 |
|
2100 |
75.9 |
|
3500 |
1.3 |
|
Exposure period 6 h, without S9 mix |
||
1% CMC▪Na |
|
100 |
Test substance |
272 |
109.6 |
454 |
93.7 |
|
756 |
102.5 |
|
1260 |
110.7 |
|
2100 |
89.8 |
|
3500 |
95.1 |
|
Exposure period 6 h, with S9 mix |
||
1% CMC▪Na |
|
100 |
Test substance |
272 |
84.3 |
454 |
85.9 |
|
756 |
85.3 |
|
1260 |
86.5 |
|
2100 |
68.3 |
|
3500 |
76.3 |
|
Table 2: Results of chromosome aberration test
Test item |
Concentration |
Aberrant cells in % |
Polyploid cells in % |
||
|
in µg/mL |
with gaps |
without gaps |
||
Exposure period 24 h, without S9 mix |
|||||
1% CMC▪Na |
|
0.5 |
0.5 |
0.0 |
|
MMC |
0.05 |
56.0 |
52.5 |
0.5 |
|
Test substance |
350 |
1.0 |
1.0 |
0.5 |
|
700 |
1.0 |
0.5 |
0.5 |
||
1400 |
0.5 |
0.0 |
0.0 |
||
2800 |
Toxic |
||||
Exposure period 48 h, without S9 mix |
|||||
1% CMC▪Na |
|
0.0 |
0.0 |
0.5 |
|
MMC |
0.025 |
58.5 |
54.5 |
0.0 |
|
Test substance |
288 |
1.0 |
1.0 |
0.0 |
|
575 |
0.5 |
0.0 |
0.5 |
||
1150 |
2.0 |
1.5 |
0.0 |
||
2300 |
Toxic |
||||
Exposure period 6 h, without S9 mix |
|||||
1% CMC▪Na |
|
1.5 |
0.5 |
0.5 |
|
CP |
12.5 |
0.5 |
0.0 |
0.5 |
|
Test substance |
875 |
0.5 |
0.5 |
0.0 |
|
1750 |
3.0 |
2.5 |
0.0 |
||
3500 |
1.0 |
0.5 |
0.5 |
||
Exposure period 6 h, with S9 mix |
|||||
1% CMC▪Na |
|
1.5 |
1.0 |
0.0 |
|
CP |
12.5 |
63.5 |
61.5 |
0.0 |
|
Test substance |
875 |
2.5 |
1.5 |
1.0 |
|
1750 |
2.0 |
2.0 |
0.0 |
||
3500 |
2.0 |
2.0 |
0.0 |
||
CMC▪Na: carboxymethylcellulose sodium (solvent)
MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)
Table 1.
Range-Finder Experiment - 3 Hour Treatment in the Absence and Presence of S-9
Concentration | 3 Hour Treatment –S‑9 | 3 Hour Treatment +S‑9 | |
µg/mL | %RS | %RS | |
0 | 100 | 100 | |
4.688 | 96 | 106 | |
9.375 | 104 | 99 | |
18.75 | 103 | 112 | |
37.5 | 98 | 108 | |
75 | 80 | 124 | |
150 | 84 | 176 | |
%RS Percent Relative Survival
Table 2.
Mutation Experiment - 3 Hour treatment in the Absence and Presence of S-9
3 Hour Treatment –S-9 | 3 Hour Treatment +S-9 | ||||
Concentration | %RS | MF § | Concentration | %RS | MF § |
µg/mL |
|
| µg/mL |
|
|
0 | 100 | 1.09 | 0 | 100 | 1.15 |
10 | 92 | 2.85 NS | 10 | 102 | 3.09 NS |
20 | 87 | 2.37 NS | 20 | 110 | 3.01 NS |
40 | 92 | 1.87 NS | 40 | 96 | 5.68# |
80 | 86 | 1.69 NS | 80 | 100 | 6.82# |
100 | 86 | 0.81 NS | 100 | 105 | 4.22 NS |
120 | 92 | 1.94 NS | 120 | 96 | 2.15 NS |
150 | 82 | 4.48# | 150 | 102 | 4.29 NS |
NQO 0.15 | 55 | 14.19 | B[a]P 2 | 66 | 8.57 |
NQO 0.2 | 43 | 25.12 | B[a]P 3 | 66 | 17.77 |
Test for Linear Trend
| -S-9 | +S-9 |
Slope | 3.22E-10 | 1.20E-08 |
Variance | 2.72E-17 | 3.58E-17 |
b² / Sb | 0.004 | 3.987 * |
Absence of S-9: Not significant
Presence of S-9: * P<0.05
%RS Percent relative survival adjusted by post treatment cell counts
NS Not significant
# Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level
*, **, *** Test for linear trend: χ 2 (one-sided), significant at 5%, 1% and 0.1% level respectively
Table 1: Dose range finding test - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay
Mean number of revertant colonies/3 replicate plates (±S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain
|
Without S9 |
With S9 |
||
Dose |
TA100 |
WP2uvrA |
TA100 |
WP2uvrA |
Positive control |
862 ± 37 |
1574 ± 37 |
1395 ± 94 |
311 ± 6 |
Solvent control |
102 ± 11 |
28 ± 6 |
82 ± 11 |
41 ± 10 |
1.7 |
88 ± 6 |
33 ± 7 |
93 ± 20 |
35 ± 8 |
5.4 |
81 ± 10 |
26 ± 6 |
105 ± 7 |
36 ± 12 |
17 |
89 ± 15 |
27 ± 9 |
102 ± 11 |
33 ± 10 |
52 |
104 ± 18 |
31 ± 4 |
104 ± 9 |
38 ± 8 |
164 |
95 ± 3 |
26 ± 7 |
105 ± 10 |
37 ± 7 |
512 |
99 ± 20 |
31 ± 1 |
95 ± 8 |
42 ± 10 |
1600 |
98 ± 16 |
33 ± 13 |
103 ± 18 |
39 ± 6 |
5000 |
87 ± 14 * |
28 ± 9 * |
93 ± 0 * |
39 ± 7 * |
* No precipitate and normal bacterial background lawn
Table 2: Experiment 1 - Mutagenic responnse in Salmonella typhimurium reverse mutation assay - Direct plate assay
Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium
|
Without S9 |
With S9 |
||||
Dose |
TA 1535 |
TA 1537 |
TA 98 |
TA 1535 |
TA 1537 |
TA 98 |
Positive control |
851 ± 29 |
421 ± 62 |
949 ± 12 |
284 ± 16 |
454 ± 42 |
1121 ± 42 |
Solvent control |
20 ± 6 |
16 ± 5 |
18 ± 2 |
15 ± 5 |
5 ± 0 |
28 ± 5 |
52 |
20 ± 10 |
8 ± 3 |
16 ± 2 |
15 ± 6 |
14 ± 6 |
39 ± 3 |
164 |
23 ± 5 |
8 ± 2 |
17 ± 8 |
12 ± 2 |
11 ± 1 |
31 ± 9 |
512 |
17± 2 |
6 ± 6 |
12 ± 3 |
17 ± 5 |
10 ± 6 |
32 ± 7 |
1600 |
18 ± 2 |
9 ± 2 |
19 ± 4 |
25 ± 2 |
10 ± 5 |
29 ± 8 |
5000 |
12 ± 0 * |
11 ± 3 * |
17 ± 4 * |
18 ± 4 * |
9 ± 4 * |
30 ± 1 * |
* No precipitate and normal bacterial background lawn
Table 3: Experiment 2 - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay - Pre-incubation assay
Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
|
Without S9 |
With S9 |
||||||||
Dose |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2uvra |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2uvra |
Positive control |
766 ± 52 |
56 ± 5 |
1003 ± 56 |
682 ± 64 |
169 ± 22 |
126 ± 9 |
160 ± 8 |
432 ± 26 |
2266 ± 183 |
399 ± 21 |
Solvent control |
17 ± 3 |
10 ± 6 |
16 ± 4 |
103 ± 13 |
33 ± 3 |
8 ± 2 |
12 ± 4 |
28 ± 3 |
115 ± 17 |
35 ± 5 |
52 |
17 ± 2 |
7 ± 3 |
19 ± 2 |
119 ± 19 |
26 ± 8 |
10 ± 5 |
13 ± 5 |
29 ± 3 |
126 ± 10 |
43 ± 16 |
164 |
16 ± 4 |
8 ± 4 |
19 ± 1 |
107 ± 10 |
27 ± 7 |
10 ± 5 |
12 ± 6 |
28 ± 3 |
110 ± 7 |
35 ± 5 |
512 |
11 ± 6 |
13 ± 2 |
15 ± 7 |
103 ± 11 |
30 ± 7 |
10 ± 5 |
9 ± 6 |
23 ± 2 |
109 ± 13 |
28 ± 5 |
1600 |
15 ± 4 |
9 ± 2 |
19 ± 7 |
103 ± 7 |
23 ± 1 |
10 ± 5 |
13 ± 11 |
33 ± 9 |
103 ± 5 |
42 ± 6 |
5000 |
17 ± 2 * |
9 ± 6 * |
18 ± 5 * |
107 ± 11 * |
29 ± 6 * |
10 ± 5 * |
15 ± 5 * |
25 ± 4 * |
112 ± 15 * |
28 ± 12 * |
* No precipitate and normal bacterial background lawn
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
The source and metal salt target substances are likely to dissociate after administration into metals ions and fatty acids due to the ionic bond between the metal cation and fatty acid ion being disrupted by polar aqueous media. The source fatty acid substance differs by chain length to that of the target but will break down to intermediate fatty acids, including glycerol, and finally carbon dioxide and water, in the same way as the target. The fatty acid component is not expected to be hazardous as it is potentially exempt from REACH under Annex V. The main difference between source and target substances is the cation and therefore test organisms would be exposed to either calcium from the target substance or lithium from the source substance. Although the cations are different, read across from lithium adipate is considered to be appropriate for in vitro gene mutation as both lithium and calcium show a similar lack of toxicity for this endpoint. The mammalian cell gene mutation assay data on the substance itself provides support to the read-across hypothesis because it provided the same result (non mutagenic) as the studies on the source substances.
Justification for classification or non-classification
Calcium dioctanoate is not classified for genetic toxicity as negative results were observed in the studies conducted on the substance itself and on valid read-across substances.
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.
