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Registration Dossier
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EC number: 701-182-0 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The three key studies involved in this assessment were in vitro
studies with aluminum, benzoate C16-18 fatty acid complexes as follows:
- gene mutation (bacterial reversion assay/Ames test; The method was
designed to conform to the guidelines for bacterial mutagenicity testing
published by the major Japanese Regulatory Authorities including METI,
MHLW and MAFF, and to meet the requirements of the OECD Guidelines for
Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method
B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS
harmonised guidelines): Negative
- Clastogenicity and aneugenicity (micronucleus assay in human
lymphocytes; The test method was designed to be compatible with OECD
Guidelines for Testing of Chemicals (2010) No 487: In Vitro
Mammalian Cell Micronucleus Test): Negative
- Mammalian cell mutagenicity (Test where mouse lymphoma-TK assay
detects the mutations at the thymidine kinase locus caused by base pair
changes, frameshift and small deletions. The test method was designed to
be compatible with OECD Guidelines for Testing of Chemicals (1997) No
476): Negative.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10 August 2012 and 07 November 2012.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- Cells: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.
The details of the donors used in this study are:
Preliminary Toxicity Test: Male, aged 27 years
Experiments 1 and 2: Female, aged 32 years
Cell Culture: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at 37ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA). - Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 fraction prepared from the liver of male Sprague-Dawley rats that had received daily doses of a mixture of phenobarbitone (80 mg/kg) and beta-naphthoflavone (100 mg/kg), prior to S9 preparation on the fourth day.
- Test concentrations with justification for top dose:
- The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:
Group
4-hour without S9 0*, 75*, 150*, 300*, 600, 900, 1200 (µg test item /mL)
4-hour with S9 (2%) 0*, 75*, 150*, 225*, 300*, 450, 600 (µg test item /mL)
20-hour without S9 0*, 75*, 150*, 300*, 600, 900, 1200 (µg test item /mL)
* = Dose level selected for analysis of binucleate cells for micronuclei - Vehicle / solvent:
- Vehicle: Minimal Essential Medium (MEM)
Justification for choice of vehicle: No data reported - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Demecolcine
- Details on test system and experimental conditions:
- Cultures incubated for 48 hours at 37ºC, in 5% CO2 in humidified air before exposure.
DURATION
- Exposure duration: 4 hours in the presence and absence of S9; also 20 hours in the absence of S9.
- Recovery time: 28 hours in presence of Cytochalasin B for all treatments.
FIXATIVE: At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375 KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4ºC prior to slide making.
STAIN: Stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
NUMBER OF CELLS EVALUATED: Proportions of mono-, bi- and multinucleate cells were evaluated for a minimum of 500 cells per culture. One thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei.
EVALUATION PROCEDURE: The micronucleus frequency in 2000 binucleated cells was analysed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data.
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity (%) = 100{(CBPIT – 1)/(CBPIC – 1)}
where:
CBPI = No. mononucleate cells + (2 x No. binucleate cells) + (3 x No. multinucleate cells)/Total number of cells
T = test chemical treatment culture
C = vehicle control culture.
OTHER DETAILS ON TEST SYSTEM:
The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Vehicle controls, test article preparations and positive controls were performed in duplicate.
For qualitative slide assessment, the slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.
From the range-finder experiment, analysising 500 cells per concentration, and using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the experiments of the main test. - Evaluation criteria:
- A toxicologically (clastogenic and aneugenic potential) significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei is less than 0.05 and there is a dose-related increase in the frequency of cells with aberrations which is reproducible.
The criteria for identifying micronuclei was that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei.
Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei can be attached by a fine nucleoplasmic bridge which is approximately no greater than one quarter of the nuclear diameter. - Statistics:
- The frequency of cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using Chi-squared Test on observed numbers of cells with micronuclei, Hoffman et al (2003).
Reference: Hoffman, WP, Garriott, ML, Lee, C. (2003). In vitro micronucleus test. In: Encyclopaedia of Biopharmaceutical Statistics, 2nd edition. S Chow ed. Marcel Dekker, Inc. New York, NY, pp. 463 – 467 - Key result
- Species / strain:
- lymphocytes:
- 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:
- not examined
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No significant change
- Effects of osmolality: ≤ 50 mOs
- Precipitation in Experiment 1: Precipitate was observed in the blood cultures at the end of the exposure period at and above 600 and 300 µg/mL in the absence and presence of S9 respectively. The precipitate was carried through onto the slides and was seen at and above 300 and 150 µg/mL in the absence and presence of S9 respectively. The maximum dose level selected for analysis of binucleate cells was limited to 300 µg/mL in both exposure groups due to the onset of precipitate affecting the cells.
- Precipitation in Experiment 2: A precipitate of the test item was observed in the blood cultures at the end of the exposure period at and above 75 µg/mL and was observed on the slides at and above 300 µg/mL. The qualitative assessment of the slides determined that there were binucleate cells up to the maximum dose tested but due to the effects of precipitate the maximum dose with binucleate cells suitable for scoring was 300 µg/mL.
RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL. The maximum dose was the maximum recommended dose level. The maximum dose level selected for the main experiments was restricted due to the onset of precipitate on the slides limiting the ability to accurately score the binucleate cells. The maximum dose selected for the 4-hour exposure groups of Experiment 1 in the absence and presence of S9 was 1200 and 600 µg/mL respectively and was 1200 µg/mL for the 20-hour exposure group in the absence of S9 in Experiment 2.
COMPARISON WITH HISTORICAL CONTROL DATA: For both experiments, the vehicle control cultures had frequencies of cells with micronuclei within the expected range (see Appendix 1 above and tables below). The positive control items induced statistically significant increases in the frequency of cells with micronuclei (see Appendix 1 above and tables below). The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. - Remarks on result:
- other: Evaluation of micronuclei in binucleate cells
- Conclusions:
- Interpretation of results Negative with metabolic activation, negative without metabolic activation
Aluminum, benzoate C16-18-fatty acids complexes did not induce statistically significant increase in the frequency of cells with micronuclei in cultured human peripheral blood lymphocytes when tested up to a limit of solubility in both the absence and presence of a rat liver metabolic activation system (S-9). The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro. - Executive summary:
Introduction
This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. The test method was designed to be compatible with, OECD Guidelines for Testing of Chemicals (2010) No 487: In Vitro Mammalian Cell Micronucleus Test.
Method
Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions were used for the study. Experiment 1 used a 4 hour exposure in the presence and absence of a standard metabolising system (S9, at a 2% final concentration). Experiment 2, used a 20-hour exposure in the absence of metabolic activation and was performed concurrently with the exposure groups of Experiment 1.
Results
All vehicle (solvent) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei, indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments. The maximum dose scored for micronuclei was limited by the onset of precipitate affecting the cell morphology and interfering with the cells on the slides.
Conclusion
The test item, aluminum, benzoate C16-18-fatty acids complexes, was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 22 August 2012 and 21 September 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- +Histidine for Salmonella.
Tryptophan for E.Coli - 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:
- phenobarbitone/betanaphthoflavone induced rat liver, S9
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Main test - Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Main test - Experiment two: 50, 150, 500, 1500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water (25 and 50 mg/mL), dimethyl sulphoxide, dimethyl formamide and acetonitrile (50 mg/mL), acetone (100 mg/mL) and tetrahydrofuran (200 mg/mL) in solubility checks performed in-house. The test item formed the best doseable suspension in sterile distilled water at 25 mg/mL; therefore, this solvent was selected as the vehicle. - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled 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 TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 10 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitroquinoline-1-oxide: 0.2 µg/plate
- Remarks:
- without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
- Details on test system and experimental conditions:
- METHODS OF APPLICATION
- Application: In agar (plate incorporation - Experiment 1, and pre-incubation - Experiment 2)
DURATION
- Preincubation period for bacterial strains: 10 hrs
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time: Not applicable
NUMBER OF REPLICATIONS: Triplicate plating.
DETERMINATION OF CYTOTOXICITY
- Method: Plates were assessed for numbers of revertant colonies using a colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 µg/plate because of excessive test item precipitation. - Evaluation criteria:
- Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.0 x 10+9 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal. - Statistics:
- Standard deviation
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test item was insoluble in sterile distilled water (25 and 50 mg/mL), dimethyl sulphoxide, dimethyl formamide and acetonitrile (50 mg/mL), acetone (100 mg/mL) and tetrahydrofuran (200 mg/mL) in solubility checks performed in-house. The test item formed the best doseable suspension in sterile distilled water at 25 mg/mL; therefore, this solvent was selected as the vehicle.
- Precipitation: A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
RANGE-FINDING/SCREENING STUDIES:
- Preliminary Toxicity Test: The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.
COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
ADDITIONAL INFORMATION ON CYTOTOXICITY: None - Conclusions:
- Interpretation of results: Negative
The test item, aluminum, aluminum, benzoate C16-18-fatty acids complexes, was considered to be non-mutagenic under the conditions of this test. - Executive summary:
Introduction.
The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.
Method.
Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item 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 (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations.
Results.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Conclusion.
The test item, aluminum, benzoate C16-18-fatty acids complexes, was considered to be non-mutagenic under the conditions of this test.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 30 January 2013 to 02 April 2013
- 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
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase, TK ±, locus of the L5178Y mouse lymphoma cell line.
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- -Type and identity of media: The stocks of cells are stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Master stocks of cells were tested and found to be free of mycoplasma.
- Periodically checked for karyotype stability: Not reported
- Periodically "cleansed" against high spontaneous background: Yes, before the stocks of cells were frozen they were cleansed of homozygous (TK ±) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital and beta-naphthoflavone induced male Sprague-Dawley liver, S9
- Test concentrations with justification for top dose:
- The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity determined in a preliminary Chromosome Aberration toxicity test.
Treatment levels:
Experiment 1
4-hour without S9 0, 4.88*, 9.77*, 19.53, 39.06, 78.13, 156.25, 312.5 and 469.75 (µg/mL)
4-hour with S9 0, 39.06*, 78.13*, 156.25, 312.5, 468.75, 625.0, 937.5 and 1250** (µg/mL)
Experiment 2
24-hour without S9 0, 5*, 10, 20, 40, 60, 80, 120 and 160* (µg/mL)
4-hour with S9 0, 40*, 80, 160, 320, 480, 640, 800** and 960** (µg/mL)
* = not plated for viability / 5-TFT resistance due to toxicity or surplus to requirements
** = treatment excluded from test statistics due to toxicity.
Overall, precipitate of the test item was aobserved at and above 10 µg/mL in the Mutagenicity Test.
Vehicle and positive controls were used in parallel with the test material. Solvent (R0 medium) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS), Sigma batches BCBH3157V and BCBG1395V at 400 µg/mL and 150 µg/mL for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0302605 at 2 µg/mL was used as the positive control in the presence of metabolic activation for experiment 1 and 2. - Vehicle / solvent:
- - Vehicle used: R0 medium treatment groups were used as the vehicle controls.
- Justification for choice of vehicle: Following solubility checks performed in-house for the Chromosome Aberration Test performed on the same test item (Harlan Laboratories. Project No. 41202616). - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- R0 medium treatment groups were used as the vehicle controls.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- R0 medium treatment groups were used as the vehicle controls.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation
- Details on test system and experimental conditions:
- This study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK ±, locus of the L5178Y mouse lymphoma cell line.
The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK ± to TK ±, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al, 1980; Jotz and Mitchell, 1981).
The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and the United Kingdom Environmental Mutagen Society. The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity). - Evaluation criteria:
- Please see "Any other information on materials and methods incl. tables" section.
- Statistics:
- Please see "Any other information on materials and methods incl. tables" section.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- non-mutagenic
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS
- Preliminary Toxicity Test: The dose range of the test material used in the Preliminary Toxicity Test was 9.77 to 2500 µg/mL. In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (% RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult. Overall, precipitate of the test item was observed at and above 19.53 μg/mL. Based on the %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item-induced toxicity.
- Mutagenicity Test in Experiment 1: There was once again evidence of marked dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values (see Table below). There was also evidence of dose related reductions in viability (%V) in both the absence and presence of metabolic activation; therefore indicating that residual toxicity had occurred. Based on the RTG and / or %RSG values, it was considered that optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. The toxicity observed at 1250 μg/mL in the presence of metabolic activation markedly exceeded the upper acceptable limit of 90% and this dose level was, therefore, excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances (see Table below). The test item induce very modest but statistically significant dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in both the absence and presence of metabolic activation, and a statistically significant increase in mutant frequency was observed at an individual dose level, the upper surviving dose level, in the presence of metabolic activation (see Table below). However, the GEF was not exceeded at any of the dose levels with acceptable levels of toxicity, and there was also no evidence of any marked increases in absolute numbers of mutant colonies. Therefore, were considered to be artefactual and of no toxicological significance. Precipitate of the test item was observed at and above 78.13 μg/mL in both the absence and presence of metabolic activation.
- Controls in Experiment 1: Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x 10(-6) viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (see Table below).
- Mutagenicity Test in Experiment 2: As was seen previously, there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the % RSG and RTG values (see Table below). Based on the % RSG and / or RTG values it was considered that optimum levels of toxicity had been achieved in both the absence and presence of metabolic activation. There was also evidence of modest dose related reductions in viability (%V) in both the absence and presence of metabolic activation; therefore indicating that residual toxicity had occurred. The excessive toxicity observed at 160 μg/mL in the absence of metabolic activation, resulted in this dose level not being plated for viability or 5-TFT resistance. The toxicity observed at and above 800 μg/mL in the presence of metabolic activation markedly exceeded the upper acceptable limit of 90% and these dose levels were, therefore, excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances (see Table below). The 24-hour exposure without metabolic activation demonstrated that the extended time point had a significant effect on the toxicity of the test item. The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10(-6) per viable cell at any of the dose levels in either the absence or presence of metabolic activation (see Table below). Precipitate of the test item observed at and above 10 μg/mL in the absence of metabolic activation, and at and above 160 μg/mL in the presence of metabolic activation.
- Controls in Experiment 2: Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x 10(-6) viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (see Table below). - Remarks on result:
- other: Strain/cell type: thymidine kinase, TK ±, locus of the L5178Y mouse lymphoma cell line
- Conclusions:
- Interpretation of results: Non-mutagenic
The test material, aluminum, benzoate C16-18-fatty acids complexes, did not induce any toxicologically significant increases in the mutant frequency at the TK ± locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test. - Executive summary:
Introduction.
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK ±, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.
Methods.
Two independent experiments were performed. In Experiment 1, L5178Y TK ± 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at up to eight dose levels, in duplicate, together with vehicle (RO medium) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at up to ten dose levels using a 4 hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation. The dose range of test material was selected following the results of a preliminary toxicity test and for Experiment 1 was 4.88 to 468.75 µg/mL in the absence of metabolic activation, and 39.06 to 1250 µg/mL in the presence of metabolic activation. The test material dose range for Experiment 2 was 5 to 160 µg/mL in the absence of metabolic activation, and 40 to 960 µg/mL in the presence of metabolic activation.
Results.
The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. Precipitate of test material was observed at and above 78.13 µg/mL in both the presence and absence of metabolic activation for Experiment 1, and at and above 10 µg/mL in the absence of metabolic activation and at and above 160 µg/mL in the presence of metabolic activation fro Experiment 2. The vehicle (RO medium) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK ± locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material, aluminum, benzoate C16-18-fatty acids complexes, did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.
Conclusion.
Aluminum, benzoate C16-18-fatty acids complexes was considered to be non-mutagenic to L5178Y cells under the conditions of the test.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Read across data
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with the Regulation (EC) No 1907/2006, Annex XI, section 1.5, read-across to aluminum, benzoate C16-18-fatty acids complexes has been used to fulfil REACH information requirements where appropriate and is justified by the chemical structures and common physiological active moieties of the substances. The chemical structures of the target and read-across substances are very closely aligned. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances. The key difference is that read-across substance contains a benzoate moiety linked to the aluminium cation, which is absent from the target substance. Benzoic acid and benzoates have been well characterized (eco)toxicologically, but in this case generating experimental data on the aluminium salt containing benzoate would be expected to demonstrate a ‘worst case’ hazard profile when compared to the target substance. Since no intrinsic toxicity could be demonstrated from any of the Annex VII or VIII endpoints with the benzoate-containing aluminium salt, then these results can be read across to the target substance without restriction.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical: Aluminum, benzoate, C16-18 fatty acids complexes (EC: 303-385-6, CAS: 94166-87-7)
See robust study summaries for further details on the identity of the tested substances and IUCLID dataset for further information on the substance identity and the data to support the read across justification.
3. ANALOGUE APPROACH JUSTIFICATION
Aluminum, benzoate C16-18-fatty acids complexes is considered suitable for read-across as it contains a fatty acid moiety coordinated to an aluminium atom. The chemical structures of the target and read-across substances are very closely aligned; both substances consist of aluminium salts of fatty acids. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances.
The fatty acids present in both substances are the same, consisting of a mixture of C16 and C18 chain lengths at approximately a 1:2 ratio. The C16 and C18 fatty acid moieties are derived from natural fatty materials, or substances which are chemically indistinguishable from natural fatty acids. The fatty acid moieties are considered not to be hazardous to humans as they are natural constituents of the human body and essential components of a balanced human nutrition. REACH Annex V, Entry 9, groups fatty acids and their potassium, sodium, calcium and magnesium salts, including C6 to C24, predominantly even-numbered, unbranched, saturated or unsaturated aliphatic monocarboxylic acids. Provided that they are obtained from natural sources and are not chemically modified, the substances included in REACH Annex V, Entry 9 are exempt from registration, unless they are classified as dangerous (except for flammability, skin irritation or eye irritation) or they meet the criteria for PBT/vPvB substances. The fatty acid components of the two substances are therefore expected to be exempt under REACH.
Fatty acids are an endogenous part of every living cell and are an essential dietary requirement. They 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 ß-oxidation uses a mitochondrial enzyme complex for a series of oxidation and hydration reactions, resulting in a cleavage of acetate groups as acetyl CoA. Acetyl CoA is used mainly to provide energy but also to provide precursors for numerous biochemical reactions. Alternative minor oxidation pathways can be found in the liver and kidney (ω-oxidation and ω-1 oxidation) and in peroxisomes for ß-methyl branched fatty acids (α-oxidation). The metabolic products can then be incorporated for example into membrane phospholipids.
Comparison of the data for the two substances indicates that they are expected to have similar properties. Neither the target or read-across substance meets the criteria for classification for physico-chemical, environmental or human health endpoints, based on the available data.
On the basis of the physico-chemical results, the substances are not flammable and have similar densities. The low vapour pressure results indicates that hazards associated with the atmospheric compartment or inhalation routes of toxicity are not expected to be relevant. The substances show similar water solubility, without surface active properties, indicating that they are likely to have similar behaviour in the aquatic environment.
Although the read-across substance met the criteria for ready biodegradability and the target substance (tested as a 50% concentration in pharmaceutical white oil) did not, neither substance was inhibitory to micro-organisms at the concentration tested. The difference in biodegradation results is expected to derive from the presence of the base oil in the target substance sample, which is designed to minimise leaching of the grease thickener, and therefore less of the grease thickener would have been available for degradation by the micro-organisms.
There are no results available for the ecotoxicity of the target substance and therefore comparison of the effect concentrations against the read-across substance is not possible. However, leaching studies on grease thickeners in base oils have been used to assess the potential bioavailability of the grease components. The bioavailability potential of the water accommodated fractions (WAFs) of metal (lithium and calcium) soap complex based grease thickeners was assessed using a solid-phase micro-extraction (SPME) method combined with gas chromatography (GC). This approach was complemented with metal ion analysis to determine whether the metal leaches out of the base grease during WAF preparation and the ecotoxicity of WAFs was also monitored using an in vitro Microtox assay. The SPME-GC data confirmed that there was negligible leaching of the thickeners from base oils in the samples tested, with measurements for calcium and lithium below the limit of detection (<0.1 mg/L) and the screening ecotoxicity data also showed a lack of toxicity of the greases.
The results of the bioavailability potential of the WAFs, the metal ion analysis and the screening ecotoxicity of lithium and calcium based complexes have been read across to aluminium based thickeners. All of these metal salts of fatty acids are expected to behave in a very similar manner when entrained within a grease matrix, with high temperature stability indicating that the thickener structure is robust and resistant to diffusion out of the oil. Dissolution of grease thickeners from grease into water is very unlikely as the thickeners are poorly water soluble and the thickeners are embedded in the hydrophobic grease matrix and thus unlikely to leach out. Therefore, although there are no data on the ecotoxicity of the target substance, no effects are expected based on the lack of bioavailability of the thickener.
These data on the potential for leaching of other metal salt complex based grease thickeners have been read across to both the target and read across substances. On the basis of these results, it is expected that neither the target nor the read across substance would leach from the base oil in which they are typically marketed and therefore neither substance would be bioavailable. Thus, reading across data from the source substance tested in its isolated form is considered robust as it provides a worst-case conclusion for the target substance which is only manufactured in an inert carrier, typically base oil. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies on the target and read-across substances themselves. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.
The available mammalian toxicity data show that neither the target nor read-across substance would be classified as irritating to skin or eyes and would not be classified for acute oral toxicity, with LD50 values of >2000 mg/kg. Although no other data are available for comparison of the potential mammalian toxicity of the two substances, the target and read-across substances are expected to behave in a very similar manner. As grease thickeners are entrained within grease matrices which are robust and resistant to diffusion out of the oil, neither substance is expected to be bioavailable. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies in fed state simulated intestinal fluid (FeSSIF) on the target and read-across substances. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.
For the genetic toxicity of the substances, read across from the source to the target substance is considered justified. Both substances would not leach when in situ in base oil during use as grease thickeners and are not expected to be bioavailable. The substances would dissociate into inorganic aluminium species and fatty acids (plus benzoic acid for the source substance), the organic components of which are readily metabolised. As the fatty acid components are essential nutrients to many organisms and are not expected to be hazardous (and the benzoate component of the source substance is not expected to be hazardous), the toxicity is expected to be driven by the aluminium component, so would be the same in both the source and target substances. As such, read across from the source substance is considered to provide a worst-case scenario for the target substance.
4. DATA
T = target substance (tests were undertaken on a sample prepared as a 50% w.w. concentration in medicinal white oil unless otherwise indicated)
RA = read-across substance
- State: Liquid (T), Solid (RA)
- Melting point: 21°C (T), 224°C (RA)
- Relative density: 0.933 (T), 1.08 (RA)
- Vapour pressure: 0.00015 Pa (T), 0.000044 Pa (RA)
- Surface tension: 72.5 mN/m (T), 72.6 mN/m (RA)
- Water solubility: ≤0.00015 g/L (T), ≤0.00026 g/L (RA)
- Flash-point: 159°C (T), No data available for RA
- Flammability: No data available for T, Not flammable (RA)
- Self-ignition temperature: 374°C (T), 383°C (RA)
- Viscosity: 174.3 mm2/s at 100°C (T), No data available for RA
- Biodegradation: Not readily biodegradable (31%) (T), Readily biodegradable (79%) (RA)
- Acute aquatic invertebrates: No data available for T, EL50 (48 h): > 100 mg/L (RA)
- Algae: No data available for T, EL50 (72 h): > 100 mg/L and NOELR (72 h): 100 mg/L (RA)
- Aquatic microorganisms: NOEC (28 d): 6.7 mg/L (T), NOEC (28 d): 15.4 mg/L (RA)
- Acute fish: No data available for T, LL50 (96 h): > 100 mg/L (RA)
- Skin irritation: Not irritating (T), Not irritating (RA)
- Eye irritation: Not classified (T), Not classified (RA)
- Skin sensitisation: No data available for T, Not sensitising (RA)
- In vitro gene mutation in bacteria: No data available for T, Negative (RA)
- Acute toxicity, oral route: LD50: > 2000 mg/kg (T, test undertaken on solid (isolated) form of the substance), LD50 >2000 mg/kg (RA)
- Acute toxicity, dermal route: No data available for T, LD50 >2000 mg/kg (RA)
- In vitro cytogenicity: No data available for T, Negative (RA)
- In vitro gene mutation in mammalian cells: No data available for T, Negative (RA)
- Short-term repeated dose toxicity, oral route: No data available for T, NOAEL: > 225 mg/kg (RA)
- Reproductive toxicity: No data available for T, NOAEL (P): > 225 mg/kg (RA)
- Developmental toxicity: No data available for T, NOAEL (F1): > 225 mg/kg (RA) - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test item was insoluble in sterile distilled water (25 and 50 mg/mL), dimethyl sulphoxide, dimethyl formamide and acetonitrile (50 mg/mL), acetone (100 mg/mL) and tetrahydrofuran (200 mg/mL) in solubility checks performed in-house. The test item formed the best doseable suspension in sterile distilled water at 25 mg/mL; therefore, this solvent was selected as the vehicle.
- Precipitation: A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
RANGE-FINDING/SCREENING STUDIES:
- Preliminary Toxicity Test: The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.
COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
ADDITIONAL INFORMATION ON CYTOTOXICITY: None - Conclusions:
- Interpretation of results: Negative
The test item, aluminum, aluminum, benzoate C16-18-fatty acids complexes, was considered to be non-mutagenic under the conditions of this test. - Executive summary:
Aluminum, benzoate C16-18-fatty acids complexes is considered suitable for read-across as it contains a fatty acid moiety coordinated to an aluminium atom. Although it also contains a coordinated benzoate ion, no toxicological effects were observed and therefore it is concluded that the benzoate ion does not contribute any additional toxicity to the substance. Aluminum, benzoate C16-18-fatty acids complexes was tested in the form of an isolated solid and showed no toxicological effects in an AMES study (Harlan 2013). Therefore, aluminium, benzoate C16 -18 fatty acids complexes is considered to non-mutagenic and this has been read across to the target substance.
Introduction.
The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.
Method.
Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item 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 (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations.
Results.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Conclusion.
The test item, aluminum, benzoate C16-18-fatty acids complexes, was considered to be non-mutagenic under the conditions of this test.
- 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:
- guideline study with acceptable restrictions
- Remarks:
- Read across data
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with the Regulation (EC) No 1907/2006, Annex XI, section 1.5, read-across to aluminum, benzoate C16-18-fatty acids complexes has been used to fulfil REACH information requirements where appropriate and is justified by the chemical structures and common physiological active moieties of the substances. The chemical structures of the target and read-across substances are very closely aligned. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances. The key difference is that read-across substance contains a benzoate moiety linked to the aluminium cation, which is absent from the target substance. Benzoic acid and benzoates have been well characterized (eco)toxicologically, but in this case generating experimental data on the aluminium salt containing benzoate would be expected to demonstrate a ‘worst case’ hazard profile when compared to the target substance. Since no intrinsic toxicity could be demonstrated from any of the Annex VII or VIII endpoints with the benzoate-containing aluminium salt, then these results can be read across to the target substance without restriction.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical: Aluminum, benzoate, C16-18 fatty acids complexes (EC: 303-385-6, CAS: 94166-87-7)
See robust study summaries for further details on the identity of the tested substances and IUCLID dataset for further information on the substance identity and the data to support the read across justification.
3. ANALOGUE APPROACH JUSTIFICATION
Aluminum, benzoate C16-18-fatty acids complexes is considered suitable for read-across as it contains a fatty acid moiety coordinated to an aluminium atom. The chemical structures of the target and read-across substances are very closely aligned; both substances consist of aluminium salts of fatty acids. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances.
The fatty acids present in both substances are the same, consisting of a mixture of C16 and C18 chain lengths at approximately a 1:2 ratio. The C16 and C18 fatty acid moieties are derived from natural fatty materials, or substances which are chemically indistinguishable from natural fatty acids. The fatty acid moieties are considered not to be hazardous to humans as they are natural constituents of the human body and essential components of a balanced human nutrition. REACH Annex V, Entry 9, groups fatty acids and their potassium, sodium, calcium and magnesium salts, including C6 to C24, predominantly even-numbered, unbranched, saturated or unsaturated aliphatic monocarboxylic acids. Provided that they are obtained from natural sources and are not chemically modified, the substances included in REACH Annex V, Entry 9 are exempt from registration, unless they are classified as dangerous (except for flammability, skin irritation or eye irritation) or they meet the criteria for PBT/vPvB substances. The fatty acid components of the two substances are therefore expected to be exempt under REACH.
Fatty acids are an endogenous part of every living cell and are an essential dietary requirement. They 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 ß-oxidation uses a mitochondrial enzyme complex for a series of oxidation and hydration reactions, resulting in a cleavage of acetate groups as acetyl CoA. Acetyl CoA is used mainly to provide energy but also to provide precursors for numerous biochemical reactions. Alternative minor oxidation pathways can be found in the liver and kidney (ω-oxidation and ω-1 oxidation) and in peroxisomes for ß-methyl branched fatty acids (α-oxidation). The metabolic products can then be incorporated for example into membrane phospholipids.
Comparison of the data for the two substances indicates that they are expected to have similar properties. Neither the target or read-across substance meets the criteria for classification for physico-chemical, environmental or human health endpoints, based on the available data.
On the basis of the physico-chemical results, the substances are not flammable and have similar densities. The low vapour pressure results indicates that hazards associated with the atmospheric compartment or inhalation routes of toxicity are not expected to be relevant. The substances show similar water solubility, without surface active properties, indicating that they are likely to have similar behaviour in the aquatic environment.
Although the read-across substance met the criteria for ready biodegradability and the target substance (tested as a 50% concentration in pharmaceutical white oil) did not, neither substance was inhibitory to micro-organisms at the concentration tested. The difference in biodegradation results is expected to derive from the presence of the base oil in the target substance sample, which is designed to minimise leaching of the grease thickener, and therefore less of the grease thickener would have been available for degradation by the micro-organisms.
There are no results available for the ecotoxicity of the target substance and therefore comparison of the effect concentrations against the read-across substance is not possible. However, leaching studies on grease thickeners in base oils have been used to assess the potential bioavailability of the grease components. The bioavailability potential of the water accommodated fractions (WAFs) of metal (lithium and calcium) soap complex based grease thickeners was assessed using a solid-phase micro-extraction (SPME) method combined with gas chromatography (GC). This approach was complemented with metal ion analysis to determine whether the metal leaches out of the base grease during WAF preparation and the ecotoxicity of WAFs was also monitored using an in vitro Microtox assay. The SPME-GC data confirmed that there was negligible leaching of the thickeners from base oils in the samples tested, with measurements for calcium and lithium below the limit of detection (<0.1 mg/L) and the screening ecotoxicity data also showed a lack of toxicity of the greases.
The results of the bioavailability potential of the WAFs, the metal ion analysis and the screening ecotoxicity of lithium and calcium based complexes have been read across to aluminium based thickeners. All of these metal salts of fatty acids are expected to behave in a very similar manner when entrained within a grease matrix, with high temperature stability indicating that the thickener structure is robust and resistant to diffusion out of the oil. Dissolution of grease thickeners from grease into water is very unlikely as the thickeners are poorly water soluble and the thickeners are embedded in the hydrophobic grease matrix and thus unlikely to leach out. Therefore, although there are no data on the ecotoxicity of the target substance, no effects are expected based on the lack of bioavailability of the thickener.
These data on the potential for leaching of other metal salt complex based grease thickeners have been read across to both the target and read across substances. On the basis of these results, it is expected that neither the target nor the read across substance would leach from the base oil in which they are typically marketed and therefore neither substance would be bioavailable. Thus, reading across data from the source substance tested in its isolated form is considered robust as it provides a worst-case conclusion for the target substance which is only manufactured in an inert carrier, typically base oil. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies on the target and read-across substances themselves. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.
The available mammalian toxicity data show that neither the target nor read-across substance would be classified as irritating to skin or eyes and would not be classified for acute oral toxicity, with LD50 values of >2000 mg/kg. Although no other data are available for comparison of the potential mammalian toxicity of the two substances, the target and read-across substances are expected to behave in a very similar manner. As grease thickeners are entrained within grease matrices which are robust and resistant to diffusion out of the oil, neither substance is expected to be bioavailable. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies in fed state simulated intestinal fluid (FeSSIF) on the target and read-across substances. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.
For the genetic toxicity of the substances, read across from the source to the target substance is considered justified. Both substances would not leach when in situ in base oil during use as grease thickeners and are not expected to be bioavailable. The substances would dissociate into inorganic aluminium species and fatty acids (plus benzoic acid for the source substance), the organic components of which are readily metabolised. As the fatty acid components are essential nutrients to many organisms and are not expected to be hazardous (and the benzoate component of the source substance is not expected to be hazardous), the toxicity is expected to be driven by the aluminium component, so would be the same in both the source and target substances. As such, read across from the source substance is considered to provide a worst-case scenario for the target substance.
4. DATA
T = target substance (tests were undertaken on a sample prepared as a 50% w.w. concentration in medicinal white oil unless otherwise indicated)
RA = read-across substance
- State: Liquid (T), Solid (RA)
- Melting point: 21°C (T), 224°C (RA)
- Relative density: 0.933 (T), 1.08 (RA)
- Vapour pressure: 0.00015 Pa (T), 0.000044 Pa (RA)
- Surface tension: 72.5 mN/m (T), 72.6 mN/m (RA)
- Water solubility: ≤0.00015 g/L (T), ≤0.00026 g/L (RA)
- Flash-point: 159°C (T), No data available for RA
- Flammability: No data available for T, Not flammable (RA)
- Self-ignition temperature: 374°C (T), 383°C (RA)
- Viscosity: 174.3 mm2/s at 100°C (T), No data available for RA
- Biodegradation: Not readily biodegradable (31%) (T), Readily biodegradable (79%) (RA)
- Acute aquatic invertebrates: No data available for T, EL50 (48 h): > 100 mg/L (RA)
- Algae: No data available for T, EL50 (72 h): > 100 mg/L and NOELR (72 h): 100 mg/L (RA)
- Aquatic microorganisms: NOEC (28 d): 6.7 mg/L (T), NOEC (28 d): 15.4 mg/L (RA)
- Acute fish: No data available for T, LL50 (96 h): > 100 mg/L (RA)
- Skin irritation: Not irritating (T), Not irritating (RA)
- Eye irritation: Not classified (T), Not classified (RA)
- Skin sensitisation: No data available for T, Not sensitising (RA)
- In vitro gene mutation in bacteria: No data available for T, Negative (RA)
- Acute toxicity, oral route: LD50: > 2000 mg/kg (T, test undertaken on solid (isolated) form of the substance), LD50 >2000 mg/kg (RA)
- Acute toxicity, dermal route: No data available for T, LD50 >2000 mg/kg (RA)
- In vitro cytogenicity: No data available for T, Negative (RA)
- In vitro gene mutation in mammalian cells: No data available for T, Negative (RA)
- Short-term repeated dose toxicity, oral route: No data available for T, NOAEL: > 225 mg/kg (RA)
- Reproductive toxicity: No data available for T, NOAEL (P): > 225 mg/kg (RA)
- Developmental toxicity: No data available for T, NOAEL (F1): > 225 mg/kg (RA) - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- non-mutagenic
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS
- Preliminary Toxicity Test: The dose range of the test material used in the Preliminary Toxicity Test was 9.77 to 2500 µg/mL. In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (% RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult. Overall, precipitate of the test item was observed at and above 19.53 μg/mL. Based on the %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item-induced toxicity.
- Mutagenicity Test in Experiment 1: There was once again evidence of marked dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values (see Table below). There was also evidence of dose related reductions in viability (%V) in both the absence and presence of metabolic activation; therefore indicating that residual toxicity had occurred. Based on the RTG and / or %RSG values, it was considered that optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. The toxicity observed at 1250 μg/mL in the presence of metabolic activation markedly exceeded the upper acceptable limit of 90% and this dose level was, therefore, excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances (see Table below). The test item induce very modest but statistically significant dose related (linear-trend) increases in the mutant frequency x 10(-6) per viable cell in both the absence and presence of metabolic activation, and a statistically significant increase in mutant frequency was observed at an individual dose level, the upper surviving dose level, in the presence of metabolic activation (see Table below). However, the GEF was not exceeded at any of the dose levels with acceptable levels of toxicity, and there was also no evidence of any marked increases in absolute numbers of mutant colonies. Therefore, these were considered to be artefactual and of no toxicological significance. Precipitate of the test item was observed at and above 78.13 μg/mL in both the absence and presence of metabolic activation.
- Controls in Experiment 1: Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x 10(-6) viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (see Table below).
- Mutagenicity Test in Experiment 2: As was seen previously, there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the % RSG and RTG values (see Table below). Based on the % RSG and / or RTG values it was considered that optimum levels of toxicity had been achieved in both the absence and presence of metabolic activation. There was also evidence of modest dose related reductions in viability (%V) in both the absence and presence of metabolic activation; therefore indicating that residual toxicity had occurred. The excessive toxicity observed at 160 μg/mL in the absence of metabolic activation, resulted in this dose level not being plated for viability or 5-TFT resistance. The toxicity observed at and above 800 μg/mL in the presence of metabolic activation markedly exceeded the upper acceptable limit of 90% and these dose levels were, therefore, excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances (see Table below). The 24-hour exposure without metabolic activation demonstrated that the extended time point had a significant effect on the toxicity of the test item. The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10(-6) per viable cell at any of the dose levels in either the absence or presence of metabolic activation (see Table below). Precipitate of the test item observed at and above 10 μg/mL in the absence of metabolic activation, and at and above 160 μg/mL in the presence of metabolic activation.
- Controls in Experiment 2: Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x 10(-6) viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (see Table below). - Remarks on result:
- other: Strain/cell type: thymidine kinase, TK ±, locus of the L5178Y mouse lymphoma cell line
- Conclusions:
- Interpretation of results: Non-mutagenic
The test material, aluminum, benzoate C16-18-fatty acids complexes, did not induce any toxicologically significant increases in the mutant frequency at the TK ± locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test. - Executive summary:
Aluminum, benzoate C16-18-fatty acids complexesis considered suitable for read-across as it contains a fatty acid moiety coordinated to an aluminium atom. Although it also contains a coordinated benzoate ion, no toxicological effects were observed and therefore it is concluded that the benzoate ion does not contribute any additional toxicity to the substance. Aluminum, benzoate C16-18-fatty acids complexes was tested in the form of an isolated solid and showed no toxicological effects in an mouse lymphoma assay study (Harlan 2013). Therefore, aluminium, benzoate C16 -18 fatty acids complexes is considered to non-mutagenic and this has been read across to the target substance.
Introduction.
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK ±, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.
Methods.
Two independent experiments were performed. In Experiment 1, L5178Y TK ± 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at up to eight dose levels, in duplicate, together with vehicle (RO medium) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at up to ten dose levels using a 4 hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation. The dose range of test material was selected following the results of a preliminary toxicity test and for Experiment 1 was 4.88 to 468.75 µg/mL in the absence of metabolic activation, and 39.06 to 1250 µg/mL in the presence of metabolic activation. The test material dose range for Experiment 2 was 5 to 160 µg/mL in the absence of metabolic activation, and 40 to 960 µg/mL in the presence of metabolic activation.
Results.
The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. Precipitate of test material was observed at and above 78.13 µg/mL in both the presence and absence of metabolic activation for Experiment 1, and at and above 10 µg/mL in the absence of metabolic activation and at and above 160 µg/mL in the presence of metabolic activation fro Experiment 2. The vehicle (RO medium) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK ± locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material, aluminum, benzoate C16-18-fatty acids complexes, did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.
Conclusion.
Aluminum, benzoate C16-18-fatty acids complexes was considered to be non-mutagenic to L5178Y cells under the conditions of the test.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- 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
- Remarks:
- Read across data
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with the Regulation (EC) No 1907/2006, Annex XI, section 1.5, read-across to aluminum, benzoate C16-18-fatty acids complexes has been used to fulfil REACH information requirements where appropriate and is justified by the chemical structures and common physiological active moieties of the substances. The chemical structures of the target and read-across substances are very closely aligned. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances. The key difference is that read-across substance contains a benzoate moiety linked to the aluminium cation, which is absent from the target substance. Benzoic acid and benzoates have been well characterized (eco)toxicologically, but in this case generating experimental data on the aluminium salt containing benzoate would be expected to demonstrate a ‘worst case’ hazard profile when compared to the target substance. Since no intrinsic toxicity could be demonstrated from any of the Annex VII or VIII endpoints with the benzoate-containing aluminium salt, then these results can be read across to the target substance without restriction.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical: Aluminum, benzoate, C16-18 fatty acids complexes (EC: 303-385-6, CAS: 94166-87-7)
See robust study summaries for further details on the identity of the tested substances and IUCLID dataset for further information on the substance identity and the data to support the read across justification.
3. ANALOGUE APPROACH JUSTIFICATION
Aluminum, benzoate C16-18-fatty acids complexes is considered suitable for read-across as it contains a fatty acid moiety coordinated to an aluminium atom. The chemical structures of the target and read-across substances are very closely aligned; both substances consist of aluminium salts of fatty acids. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances.
The fatty acids present in both substances are the same, consisting of a mixture of C16 and C18 chain lengths at approximately a 1:2 ratio. The C16 and C18 fatty acid moieties are derived from natural fatty materials, or substances which are chemically indistinguishable from natural fatty acids. The fatty acid moieties are considered not to be hazardous to humans as they are natural constituents of the human body and essential components of a balanced human nutrition. REACH Annex V, Entry 9, groups fatty acids and their potassium, sodium, calcium and magnesium salts, including C6 to C24, predominantly even-numbered, unbranched, saturated or unsaturated aliphatic monocarboxylic acids. Provided that they are obtained from natural sources and are not chemically modified, the substances included in REACH Annex V, Entry 9 are exempt from registration, unless they are classified as dangerous (except for flammability, skin irritation or eye irritation) or they meet the criteria for PBT/vPvB substances. The fatty acid components of the two substances are therefore expected to be exempt under REACH.
Fatty acids are an endogenous part of every living cell and are an essential dietary requirement. They 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 ß-oxidation uses a mitochondrial enzyme complex for a series of oxidation and hydration reactions, resulting in a cleavage of acetate groups as acetyl CoA. Acetyl CoA is used mainly to provide energy but also to provide precursors for numerous biochemical reactions. Alternative minor oxidation pathways can be found in the liver and kidney (ω-oxidation and ω-1 oxidation) and in peroxisomes for ß-methyl branched fatty acids (α-oxidation). The metabolic products can then be incorporated for example into membrane phospholipids.
Comparison of the data for the two substances indicates that they are expected to have similar properties. Neither the target or read-across substance meets the criteria for classification for physico-chemical, environmental or human health endpoints, based on the available data.
On the basis of the physico-chemical results, the substances are not flammable and have similar densities. The low vapour pressure results indicates that hazards associated with the atmospheric compartment or inhalation routes of toxicity are not expected to be relevant. The substances show similar water solubility, without surface active properties, indicating that they are likely to have similar behaviour in the aquatic environment.
Although the read-across substance met the criteria for ready biodegradability and the target substance (tested as a 50% concentration in pharmaceutical white oil) did not, neither substance was inhibitory to micro-organisms at the concentration tested. The difference in biodegradation results is expected to derive from the presence of the base oil in the target substance sample, which is designed to minimise leaching of the grease thickener, and therefore less of the grease thickener would have been available for degradation by the micro-organisms.
There are no results available for the ecotoxicity of the target substance and therefore comparison of the effect concentrations against the read-across substance is not possible. However, leaching studies on grease thickeners in base oils have been used to assess the potential bioavailability of the grease components. The bioavailability potential of the water accommodated fractions (WAFs) of metal (lithium and calcium) soap complex based grease thickeners was assessed using a solid-phase micro-extraction (SPME) method combined with gas chromatography (GC). This approach was complemented with metal ion analysis to determine whether the metal leaches out of the base grease during WAF preparation and the ecotoxicity of WAFs was also monitored using an in vitro Microtox assay. The SPME-GC data confirmed that there was negligible leaching of the thickeners from base oils in the samples tested, with measurements for calcium and lithium below the limit of detection (<0.1 mg/L) and the screening ecotoxicity data also showed a lack of toxicity of the greases.
The results of the bioavailability potential of the WAFs, the metal ion analysis and the screening ecotoxicity of lithium and calcium based complexes have been read across to aluminium based thickeners. All of these metal salts of fatty acids are expected to behave in a very similar manner when entrained within a grease matrix, with high temperature stability indicating that the thickener structure is robust and resistant to diffusion out of the oil. Dissolution of grease thickeners from grease into water is very unlikely as the thickeners are poorly water soluble and the thickeners are embedded in the hydrophobic grease matrix and thus unlikely to leach out. Therefore, although there are no data on the ecotoxicity of the target substance, no effects are expected based on the lack of bioavailability of the thickener.
These data on the potential for leaching of other metal salt complex based grease thickeners have been read across to both the target and read across substances. On the basis of these results, it is expected that neither the target nor the read across substance would leach from the base oil in which they are typically marketed and therefore neither substance would be bioavailable. Thus, reading across data from the source substance tested in its isolated form is considered robust as it provides a worst-case conclusion for the target substance which is only manufactured in an inert carrier, typically base oil. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies on the target and read-across substances themselves. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.
The available mammalian toxicity data show that neither the target nor read-across substance would be classified as irritating to skin or eyes and would not be classified for acute oral toxicity, with LD50 values of >2000 mg/kg. Although no other data are available for comparison of the potential mammalian toxicity of the two substances, the target and read-across substances are expected to behave in a very similar manner. As grease thickeners are entrained within grease matrices which are robust and resistant to diffusion out of the oil, neither substance is expected to be bioavailable. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies in fed state simulated intestinal fluid (FeSSIF) on the target and read-across substances. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.
For the genetic toxicity of the substances, read across from the source to the target substance is considered justified. Both substances would not leach when in situ in base oil during use as grease thickeners and are not expected to be bioavailable. The substances would dissociate into inorganic aluminium species and fatty acids (plus benzoic acid for the source substance), the organic components of which are readily metabolised. As the fatty acid components are essential nutrients to many organisms and are not expected to be hazardous (and the benzoate component of the source substance is not expected to be hazardous), the toxicity is expected to be driven by the aluminium component, so would be the same in both the source and target substances. As such, read across from the source substance is considered to provide a worst-case scenario for the target substance.
4. DATA
T = target substance (tests were undertaken on a sample prepared as a 50% w.w. concentration in medicinal white oil unless otherwise indicated)
RA = read-across substance
- State: Liquid (T), Solid (RA)
- Melting point: 21°C (T), 224°C (RA)
- Relative density: 0.933 (T), 1.08 (RA)
- Vapour pressure: 0.00015 Pa (T), 0.000044 Pa (RA)
- Surface tension: 72.5 mN/m (T), 72.6 mN/m (RA)
- Water solubility: ≤0.00015 g/L (T), ≤0.00026 g/L (RA)
- Flash-point: 159°C (T), No data available for RA
- Flammability: No data available for T, Not flammable (RA)
- Self-ignition temperature: 374°C (T), 383°C (RA)
- Viscosity: 174.3 mm2/s at 100°C (T), No data available for RA
- Biodegradation: Not readily biodegradable (31%) (T), Readily biodegradable (79%) (RA)
- Acute aquatic invertebrates: No data available for T, EL50 (48 h): > 100 mg/L (RA)
- Algae: No data available for T, EL50 (72 h): > 100 mg/L and NOELR (72 h): 100 mg/L (RA)
- Aquatic microorganisms: NOEC (28 d): 6.7 mg/L (T), NOEC (28 d): 15.4 mg/L (RA)
- Acute fish: No data available for T, LL50 (96 h): > 100 mg/L (RA)
- Skin irritation: Not irritating (T), Not irritating (RA)
- Eye irritation: Not classified (T), Not classified (RA)
- Skin sensitisation: No data available for T, Not sensitising (RA)
- In vitro gene mutation in bacteria: No data available for T, Negative (RA)
- Acute toxicity, oral route: LD50: > 2000 mg/kg (T, test undertaken on solid (isolated) form of the substance), LD50 >2000 mg/kg (RA)
- Acute toxicity, dermal route: No data available for T, LD50 >2000 mg/kg (RA)
- In vitro cytogenicity: No data available for T, Negative (RA)
- In vitro gene mutation in mammalian cells: No data available for T, Negative (RA)
- Short-term repeated dose toxicity, oral route: No data available for T, NOAEL: > 225 mg/kg (RA)
- Reproductive toxicity: No data available for T, NOAEL (P): > 225 mg/kg (RA)
- Developmental toxicity: No data available for T, NOAEL (F1): > 225 mg/kg (RA) - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- lymphocytes:
- 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:
- not examined
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No significant change
- Effects of osmolality: ≤ 50 mOs
- Precipitation in Experiment 1: Precipitate was observed in the blood cultures at the end of the exposure period at and above 600 and 300 µg/mL in the absence and presence of S9 respectively. The precipitate was carried through onto the slides and was seen at and above 300 and 150 µg/mL in the absence and presence of S9 respectively. The maximum dose level selected for analysis of binucleate cells was limited to 300 µg/mL in both exposure groups due to the onset of precipitate affecting the cells.
- Precipitation in Experiment 2: A precipitate of the test item was observed in the blood cultures at the end of the exposure period at and above 75 µg/mL and was observed on the slides at and above 300 µg/mL. The qualitative assessment of the slides determined that there were binucleate cells up to the maximum dose tested but due to the effects of precipitate the maximum dose with binucleate cells suitable for scoring was 300 µg/mL.
RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL. The maximum dose was the maximum recommended dose level. The maximum dose level selected for the main experiments was restricted due to the onset of precipitate on the slides limiting the ability to accurately score the binucleate cells. The maximum dose selected for the 4-hour exposure groups of Experiment 1 in the absence and presence of S9 was 1200 and 600 µg/mL respectively and was 1200 µg/mL for the 20-hour exposure group in the absence of S9 in Experiment 2.
COMPARISON WITH HISTORICAL CONTROL DATA: For both experiments, the vehicle control cultures had frequencies of cells with micronuclei within the expected range (see Appendix 1 above and tables below). The positive control items induced statistically significant increases in the frequency of cells with micronuclei (see Appendix 1 above and tables below). The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. - Remarks on result:
- other: Evaluation of micronuclei in binucleate cells
- Conclusions:
- Interpretation of results: Negative with metabolic activation, negative without metabolic activation
Aluminum, benzoate C16-18-fatty acids complexes did not induce statistically significant increase in the frequency of cells with micronuclei in cultured human peripheral blood lymphocytes when tested up to a limit of solubility in both the absence and presence of a rat liver metabolic activation system (S-9). The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro. - Executive summary:
Aluminum, benzoate C16-18-fatty acids complexesis considered suitable for read-across as it contains a fatty acid moiety coordinated to an aluminium atom. Although it also contains a coordinated benzoate ion, no toxicological effects were observed and therefore it is concluded that the benzoate ion does not contribute any additional toxicity to the substance. Aluminum, benzoate C16-18-fatty acids complexes was tested in the form of an isolated solid and showed no toxicological effects in an micronucleus study (Harlan 2013). Therefore, aluminium, benzoate C16 -18 fatty acids complexes is considered to non-clastogenic and non-aneugenic and this has been read across to the target substance.
Introduction
This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. The test method was designed to be compatible with, OECD Guidelines for Testing of Chemicals (2010) No 487: In Vitro Mammalian Cell Micronucleus Test.
Method
Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions were used for the study. Experiment 1 used a 4 hour exposure in the presence and absence of a standard metabolising system (S9, at a 2% final concentration). Experiment 2, used a 20-hour exposure in the absence of metabolic activation and was performed concurrently with the exposure groups of Experiment 1.
Results
All vehicle (solvent) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei, indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments. The maximum dose scored for micronuclei was limited by the onset of precipitate affecting the cell morphology and interfering with the cells on the slides.
Conclusion
The test item, aluminum, benzoate C16-18-fatty acids complexes, was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
Referenceopen allclose all
Table 2. CBPI – Experiment 1
4-Hour exposure without S9 |
4-Hour exposure without S9 (2%) |
||||||||||||||
Dose level (µg/ml) |
Replicate |
Nucleate cells/500 cells |
CBPI |
Mean CBPI |
% control CBPI |
Dose level (µg/ml) |
Replicate |
Nucleate cells/500 cells |
CBPI |
Mean CBPI |
% control CBPI |
||||
Mono |
Bi |
Multi |
Mono |
Bi |
Multi |
||||||||||
0 |
A |
190 |
230 |
80 |
1.78 |
1.58 |
0 |
0 |
A |
126 |
254 |
120 |
1.99 |
1.96 |
100 |
B |
344 |
118 |
38 |
1.39 |
B |
130 |
275 |
95 |
1.93 |
||||||
75 |
A |
167 |
231 |
102 |
1.87 |
1.66 |
1.12 |
75 |
A |
264 |
180 |
56 |
1.58 |
1.79 |
82 |
B |
311 |
158 |
31 |
1.44 |
B |
118 |
266 |
116 |
2.00 |
||||||
150 |
A |
168 |
261 |
71 |
1.81 |
1.73 |
125 |
150* |
A |
327 |
150 |
23 |
1.39 |
1.68 |
71 |
B |
219 |
234 |
47 |
1.68 |
B |
132 |
252 |
116 |
1.97 |
||||||
300† |
A |
219 |
240 |
41 |
1.64 |
1.58 |
99 |
225* |
A |
209 |
253 |
38 |
1.66 |
1.80 |
83 |
B |
261 |
219 |
20 |
1.52 |
B |
122 |
287 |
91 |
1.94 |
||||||
600P† |
A |
NSB |
NSB |
NSB |
NSB. |
NSB |
NSB |
300P* |
A |
194 |
226 |
80 |
1.77 |
1.79 |
82 |
B |
NSB |
NSB |
NSB |
NSB. |
B |
162 |
276 |
62 |
1.80 |
||||||
900P† |
A |
NB |
NB |
NB |
NB |
NB |
NB |
450P* |
NSB |
NSB |
NSB |
NSB. |
NSB |
NSB |
NSB |
B |
NB |
NB |
NB |
NB |
NSB |
NSB |
NSB |
NSB. |
NSB |
||||||
1200P† |
A |
NB |
NB |
NB |
NB |
NB |
NB |
600P* |
NSB |
NSB |
NSB |
NSB. |
NSB |
NSB |
NSB |
B |
NB |
NB |
NB |
NB |
NSB |
NSB |
NSB |
NSB. |
NSB |
||||||
MMC 0.2 |
A |
244 |
239 |
17 |
1.55 |
1.54 |
92 |
CP 5 |
A |
260 |
234 |
6 |
1.49 |
1.48 |
50 |
B |
248 |
237 |
15 |
1.53 |
B |
274 |
219 |
7 |
1.47 |
||||||
MMC = Mitomycin C
CP = Cyclophosphamide
NB = No binucleate cells
NSB = No binucleate cells suitable for scoring
P = Precipitate observed at the end of exposure period
† = Precipitate observed on the slides
Table 3: CBPI – Experiment 2
20-Hour exposure without S9 |
|||||||
Dose level (µg/ml) |
Replicate |
Nucleate cells/500 cells |
CBPI |
Mean CBPI |
% Control CBPI |
||
Mono |
Bi |
Multi |
|||||
0 |
A |
48 |
326 |
126 |
2.16 |
2.20 |
100 |
B |
41 |
300 |
159 |
2.25 |
|||
75P |
A |
52 |
328 |
120 |
2.14 |
2.07 |
89 |
B |
87 |
325 |
88 |
2.00 |
|||
150P |
A |
83 |
314 |
103 |
2.04 |
2.11 |
93 |
B |
41 |
325 |
134 |
2.19 |
|||
300P† |
A |
129 |
299 |
72 |
1.89 |
1.87 |
73 |
B |
158 |
259 |
83 |
1.85 |
|||
600P† |
A |
NSB |
NSB |
NSB |
NSB |
NSB |
NSB |
B |
NSB |
NSB |
NSB |
NSB |
|||
900P† |
A |
NSB |
NSB |
NSB |
NSB |
NSB |
NSB |
B |
NSB |
NSB |
NSB |
NSB |
|||
1200P† |
A |
NSB |
NSB |
NSB |
NSB |
NSB |
NSB |
B |
NSB |
NSB |
NSB |
NSB |
|||
DC 0.075 |
A |
251 |
206 |
43 |
1.58 |
1.58 |
48 |
B |
249 |
214 |
37 |
1.58 |
|||
DC = Demecolcine
NSB = No binucleate cells suitable for scoring
P = Precipitate observed at the end of exposure period
† = Precipitate observed on the slides
RESULTS
Preliminary ToxicityTest
The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.
The numbers of revertant colonies for the toxicity assay were:
With (+) or without (-) S9-mix |
Strain |
Dose (µg/plate) |
||||||||||
0 |
0.15 |
0.5 |
1.5 |
5 |
15 |
50 |
150 |
500 |
1500 |
5000 |
||
- |
TA100 |
89 |
84 |
85 |
98 |
91 |
98 |
94 |
94 |
116 |
99P |
81P |
+ |
TA100 |
89 |
95 |
85 |
93 |
87 |
73 |
110 |
94 |
102 |
88 |
91P |
- |
WP2uvrA |
24 |
31 |
28 |
27 |
18 |
21 |
30 |
19 |
29 |
31 |
39P |
+ |
WP2uvrA |
29 |
21 |
31 |
37 |
24 |
19 |
36 |
25 |
35 |
33P |
33P |
P: Precipitate
MutationTest
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) are presented in Table1 (see below) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.
No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.
All
of the positive control chemicals used in the test induced
marked increases in the frequency of revertant colonies thus
confirming the activity of the S9-mix and the sensitivity of
the bacterial strains.
Table1 Spontaneous Mutation Rates (Concurrent Negative Controls)
Experiment 1
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
84 |
|
18 |
|
26 |
|
14 |
|
7 |
|
66 |
(77) |
12 |
(15) |
23 |
(23) |
19 |
(15) |
15 |
(10) |
81 |
|
14 |
|
21 |
|
13 |
|
9 |
|
Experiment 2
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
93 |
|
29 |
|
18 |
|
12 |
|
11 |
|
103 |
(100) |
18 |
(25) |
21 |
(24) |
18 |
(18) |
10 |
(14) |
103 |
|
27 |
|
32 |
|
25 |
|
20 |
|
Table 2: Test Results: Experiment 1– Without Metabolic Activation
With or without S9-Mix |
Dose Level Per Plate |
Number of revertants (mean) ± SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
|||||||
S9-Mix (-) |
Solvent Control (Water) |
101 |
(90) 16.8# |
23 |
(190) 4.6 |
23 |
(26) 4.2 |
20 |
(22) 3.2 |
9 |
(9) 0.6 |
99 |
20 |
31 |
26 |
10 |
|||||||
71 |
14 |
25 |
21 |
9 |
|||||||
50 μg |
90 |
(85) 10.8 |
11 |
(16) 4.5 |
26 |
(20) 5.1 |
12 |
(21) 10.7 |
10 |
(13) 2.5 |
|
93 |
16 |
16 |
33 |
15 |
|||||||
73 |
20 |
19 |
19 |
13 |
|||||||
150 μg |
103 |
(81) 19.1 |
19 |
(18) 2.3 |
29 |
(25) 4.0 |
15 |
(17) 2.9 |
12 |
(12) 2.5 |
|
70 |
19 |
24 |
20 |
14 |
|||||||
70 |
15 |
21 |
15 |
9 |
|||||||
500 μg |
113 |
(103) 9.1 |
20 |
(20) 2.5 |
38 |
(31) 5.9 |
20 |
(21) 0.6 |
9 |
(10) 2.3 |
|
95 |
18 |
29 |
21 |
9 |
|||||||
102 |
23 |
27 |
21 |
13 |
|||||||
1500 μg |
106p |
(95) 10.6 |
24P |
(21) 4.2 |
25P |
(28) 4.6 |
19P |
(24) 6.4 |
9P |
(9) 2.5 |
|
93P |
16P |
25P |
31P |
12P |
|||||||
85P |
22P |
33P |
21P |
7P |
|||||||
5000 μg |
98P |
(86) 10.6 |
20P |
(21) 1.5 |
21P |
(24) 4.9 |
24p |
(24) 4.9 |
9P |
(11) 1.5 |
|
78P |
21P |
30P |
25P |
11P |
|||||||
82P |
23P |
22P |
23P |
12P |
|||||||
Positive controls S9-Mix (-) |
Name |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||
Concentration (μg/plate) |
3 μg |
5 μg |
2 μg |
0.2 μg |
80 μg |
||||||
No. colonies per plate |
577 |
(560) 48.1 |
284 |
(304) 63.4 |
977 |
(807) 147.5 |
103 |
(107) 3.2 |
1054 |
(94.9) 183.9 |
|
629 |
253 |
713 |
108 |
737 |
|||||||
533 |
375 |
731 |
109 |
1057 |
|||||||
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
P Precipitate
# Standard deviation
Table 3 Test Results: Experiment 1 – With Metabolic Activation
With or without S9-Mix |
Dose Level Per Plate |
Number of revertants (mean) ± SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
|||||||
S9-Mix (+) |
Solvent Control (Water) |
106 |
(108) 11.6# |
12 |
(130)2.3 |
35 |
(38) 7.4 |
22 |
(25) 4.6 |
14 |
(12) 2.0 |
97 |
12 |
32 |
22 |
10 |
|||||||
120 |
16 |
46 |
30 |
12 |
|||||||
50 μg |
101 |
(96) 13.6 |
14 |
914) 0.6 |
38 |
(32) 10.1 |
29 |
(27) 2.5 |
13 |
(13) 1.5 |
|
107 |
14 |
37 |
27 |
15 |
|||||||
81 |
15 |
20 |
24 |
12 |
|||||||
150 μg |
111 |
(108) 14.2 |
14 |
(14) 0.6 |
34 |
(39) 6.4 |
23 |
(27) 4.7 |
12 |
(12) 3.0 |
|
121 |
14 |
46 |
25 |
15 |
|||||||
93 |
13 |
36 |
32 |
9 |
|||||||
500 μg |
118 |
(108) 20.3 |
19 |
(15) 5.1 |
29 |
(34) 5.7 |
14 |
(23) 8.3 |
11 |
(10) 3.1 |
|
122 |
16 |
32 |
26 |
13 |
|||||||
85 |
9 |
40 |
30 |
7 |
|||||||
1500 μg |
113P |
(111) 6.7 |
10p |
(12) 1.5 |
31P |
(32) 2.6 |
23P |
(26) 5.2 |
12P |
(12) 0.6 |
|
117P |
13P |
35P |
23P |
13P |
|||||||
104P |
12P |
30P |
32P |
12P |
|||||||
5000 μg |
113P |
(113) 3.0 |
12P |
(13) 4.6 |
36P |
(38) 5.3 |
31P |
(270) 4.0 |
7p |
(10) 3.0 |
|
110P |
9P |
34P |
23P |
13P |
|||||||
116P |
18P |
44P |
26P |
10P |
|||||||
Positive controls S9-Mix (+) |
Name |
2AA |
2AA |
2AA |
BP |
2AA |
|||||
Concentration (μg/plate) |
1 μg |
2 μg |
10 μg |
5 μg |
2 μg |
||||||
No. colonies per plate |
827 |
(817) 10.5 |
167 |
(161) 6.0 |
400 |
(400) 10.5 |
207 |
(246) 34.1 |
203 |
(212) 15.6 |
|
806 |
155 |
390 |
268 |
230 |
|||||||
818 |
162 |
411 |
264 |
203 |
|||||||
2AA 2-Aminoanthracene
BP Benzo(a)pyrene
P Precipitate
# Standard deviation
Tables 4 and 5 below
Table: Summary of the Mouse Lymphoma Assay on Aluminum, benzoate C16-18-fatty acids complexes (41205678)
Experiment 1
Treatment (µg/ml) |
4-Hours-S9 |
Treatment (µg/ml) |
4-Hours+S9 |
||||||||||
|
%RSG |
RTG |
MF§ |
|
%RSG |
RTG |
MF§ |
||||||
0 |
|
100 |
1.00 |
123.96 |
|
0 |
|
100 |
1.00 |
128.09 |
|
||
4.88 |
Ø |
91 |
|
|
|
39.06 |
Ø |
107 |
|
|
|
||
9.77 |
Ø |
92 |
|
|
|
78.13 |
Ø |
100 |
|
|
|
||
19.53 |
|
90 |
0.83 |
129.32 |
|
156.25 |
|
89 |
0.75 |
141.53 |
|
||
39.06 |
|
84 |
0.91 |
134.73 |
|
312.5 |
|
69 |
0.51 |
120.70 |
|
||
78.13 |
|
81 |
0.72 |
116.77 |
|
468.75 |
|
64 |
0.44 |
136.24 |
|
||
156.25 |
|
64 |
0.61 |
143.60 |
|
625 |
|
46 |
0.29 |
146.90 |
|
||
312.5 |
|
42 |
0.37 |
153.18 |
|
937.5 |
|
20 |
0.08 |
196.66 |
* |
||
468.75 |
|
21 |
0.12 |
160.99 |
|
1250 |
X |
13 |
0.02 |
369.05 |
|
||
Linear trend |
|
* |
Linear trend |
|
* |
||||||||
EMS |
|
|
|
|
|
CP |
|
|
|
|
|
||
400 |
|
60 |
0.46 |
1022.04 |
|
2 |
|
55 |
0.24 |
1459.85 |
|
||
|
|
|
|
|
|
|
|
|
|
|
|
||
Experiment 2
Treatment (µg/ml) |
24-Hours-S9 |
Treatment (µg/ml) |
4-Hours+S9 |
||||||||
|
%RSG |
RTG |
MF§ |
|
%RSG |
RTG |
MF§ |
||||
0 |
|
100 |
1.00 |
108.39 |
|
0 |
|
100 |
1.00 |
110.11 |
|
5 |
Ø |
95 |
|
|
|
40 |
Ø |
96 |
|
|
|
10 |
|
96 |
0.77 |
103.57 |
|
80 |
|
90 |
0.97 |
119.73 |
|
20 |
|
73 |
0.78 |
105.22 |
|
160 |
|
70 |
0.82 |
116.68 |
|
40 |
|
46 |
0.58 |
127.28 |
|
320 |
|
38 |
0.30 |
143.02 |
|
60 |
|
42 |
0.58 |
105.28 |
|
480 |
|
37 |
0.30 |
128.14 |
|
80 |
|
16 |
0.26 |
126.27 |
|
640 |
|
29 |
0.22 |
123.00 |
|
120 |
|
10 |
0.16 |
128.14 |
|
800 |
X |
19 |
0.05 |
298.62 |
|
160 |
Ø |
2 |
|
|
|
960 |
X |
13 |
0.02 |
293.81 |
|
Linear trend |
|
NS |
Linear trend |
|
NS |
||||||
EMS |
|
|
|
|
|
CP |
|
|
|
|
|
150 |
|
38 |
0.33 |
1112.59 |
|
2 |
|
55 |
0.30 |
767.84 |
|
MF§ 5 -TFT resistant mutants/106viable cells 2 days after treatment
% Percent relative survival adjusted by post treatment cell count factor
RTG Relative total growth adjusted to account for immediate post-treatment toxicity
Ø Not plated for viability / 5-TFT resistance due to toxicity or surplus to requirements
X Treatment excluded from test statistics due to toxicity
* Significant at 5%
NS Not significant
RESULTS
Preliminary ToxicityTest
The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.
The numbers of revertant colonies for the toxicity assay were:
With (+) or without (-) S9-mix |
Strain |
Dose (µg/plate) |
||||||||||
0 |
0.15 |
0.5 |
1.5 |
5 |
15 |
50 |
150 |
500 |
1500 |
5000 |
||
- |
TA100 |
89 |
84 |
85 |
98 |
91 |
98 |
94 |
94 |
116 |
99P |
81P |
+ |
TA100 |
89 |
95 |
85 |
93 |
87 |
73 |
110 |
94 |
102 |
88 |
91P |
- |
WP2uvrA |
24 |
31 |
28 |
27 |
18 |
21 |
30 |
19 |
29 |
31 |
39P |
+ |
WP2uvrA |
29 |
21 |
31 |
37 |
24 |
19 |
36 |
25 |
35 |
33P |
33P |
P: Precipitate
MutationTest
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) are presented in Table1 (see below) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.
No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.
All
of the positive control chemicals used in the test induced
marked increases in the frequency of revertant colonies thus
confirming the activity of the S9-mix and the sensitivity of
the bacterial strains.
Table1 Spontaneous Mutation Rates (Concurrent Negative Controls)
Experiment 1
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
84 |
|
18 |
|
26 |
|
14 |
|
7 |
|
66 |
(77) |
12 |
(15) |
23 |
(23) |
19 |
(15) |
15 |
(10) |
81 |
|
14 |
|
21 |
|
13 |
|
9 |
|
Experiment 2
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
93 |
|
29 |
|
18 |
|
12 |
|
11 |
|
103 |
(100) |
18 |
(25) |
21 |
(24) |
18 |
(18) |
10 |
(14) |
103 |
|
27 |
|
32 |
|
25 |
|
20 |
|
Table 2: Test Results: Experiment 1– Without Metabolic Activation
With or without S9-Mix |
Dose Level Per Plate |
Number of revertants (mean) ± SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
|||||||
S9-Mix (-) |
Solvent Control (Water) |
101 |
(90) 16.8# |
23 |
(190) 4.6 |
23 |
(26) 4.2 |
20 |
(22) 3.2 |
9 |
(9) 0.6 |
99 |
20 |
31 |
26 |
10 |
|||||||
71 |
14 |
25 |
21 |
9 |
|||||||
50 μg |
90 |
(85) 10.8 |
11 |
(16) 4.5 |
26 |
(20) 5.1 |
12 |
(21) 10.7 |
10 |
(13) 2.5 |
|
93 |
16 |
16 |
33 |
15 |
|||||||
73 |
20 |
19 |
19 |
13 |
|||||||
150 μg |
103 |
(81) 19.1 |
19 |
(18) 2.3 |
29 |
(25) 4.0 |
15 |
(17) 2.9 |
12 |
(12) 2.5 |
|
70 |
19 |
24 |
20 |
14 |
|||||||
70 |
15 |
21 |
15 |
9 |
|||||||
500 μg |
113 |
(103) 9.1 |
20 |
(20) 2.5 |
38 |
(31) 5.9 |
20 |
(21) 0.6 |
9 |
(10) 2.3 |
|
95 |
18 |
29 |
21 |
9 |
|||||||
102 |
23 |
27 |
21 |
13 |
|||||||
1500 μg |
106p |
(95) 10.6 |
24P |
(21) 4.2 |
25P |
(28) 4.6 |
19P |
(24) 6.4 |
9P |
(9) 2.5 |
|
93P |
16P |
25P |
31P |
12P |
|||||||
85P |
22P |
33P |
21P |
7P |
|||||||
5000 μg |
98P |
(86) 10.6 |
20P |
(21) 1.5 |
21P |
(24) 4.9 |
24p |
(24) 4.9 |
9P |
(11) 1.5 |
|
78P |
21P |
30P |
25P |
11P |
|||||||
82P |
23P |
22P |
23P |
12P |
|||||||
Positive controls S9-Mix (-) |
Name |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||
Concentration (μg/plate) |
3 μg |
5 μg |
2 μg |
0.2 μg |
80 μg |
||||||
No. colonies per plate |
577 |
(560) 48.1 |
284 |
(304) 63.4 |
977 |
(807) 147.5 |
103 |
(107) 3.2 |
1054 |
(94.9) 183.9 |
|
629 |
253 |
713 |
108 |
737 |
|||||||
533 |
375 |
731 |
109 |
1057 |
|||||||
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
P Precipitate
# Standard deviation
Table 3 Test Results: Experiment 1 – With Metabolic Activation
With or without S9-Mix |
Dose Level Per Plate |
Number of revertants (mean) ± SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
|||||||
S9-Mix (+) |
Solvent Control (Water) |
106 |
(108) 11.6# |
12 |
(130)2.3 |
35 |
(38) 7.4 |
22 |
(25) 4.6 |
14 |
(12) 2.0 |
97 |
12 |
32 |
22 |
10 |
|||||||
120 |
16 |
46 |
30 |
12 |
|||||||
50 μg |
101 |
(96) 13.6 |
14 |
914) 0.6 |
38 |
(32) 10.1 |
29 |
(27) 2.5 |
13 |
(13) 1.5 |
|
107 |
14 |
37 |
27 |
15 |
|||||||
81 |
15 |
20 |
24 |
12 |
|||||||
150 μg |
111 |
(108) 14.2 |
14 |
(14) 0.6 |
34 |
(39) 6.4 |
23 |
(27) 4.7 |
12 |
(12) 3.0 |
|
121 |
14 |
46 |
25 |
15 |
|||||||
93 |
13 |
36 |
32 |
9 |
|||||||
500 μg |
118 |
(108) 20.3 |
19 |
(15) 5.1 |
29 |
(34) 5.7 |
14 |
(23) 8.3 |
11 |
(10) 3.1 |
|
122 |
16 |
32 |
26 |
13 |
|||||||
85 |
9 |
40 |
30 |
7 |
|||||||
1500 μg |
113P |
(111) 6.7 |
10p |
(12) 1.5 |
31P |
(32) 2.6 |
23P |
(26) 5.2 |
12P |
(12) 0.6 |
|
117P |
13P |
35P |
23P |
13P |
|||||||
104P |
12P |
30P |
32P |
12P |
|||||||
5000 μg |
113P |
(113) 3.0 |
12P |
(13) 4.6 |
36P |
(38) 5.3 |
31P |
(270) 4.0 |
7p |
(10) 3.0 |
|
110P |
9P |
34P |
23P |
13P |
|||||||
116P |
18P |
44P |
26P |
10P |
|||||||
Positive controls S9-Mix (+) |
Name |
2AA |
2AA |
2AA |
BP |
2AA |
|||||
Concentration (μg/plate) |
1 μg |
2 μg |
10 μg |
5 μg |
2 μg |
||||||
No. colonies per plate |
827 |
(817) 10.5 |
167 |
(161) 6.0 |
400 |
(400) 10.5 |
207 |
(246) 34.1 |
203 |
(212) 15.6 |
|
806 |
155 |
390 |
268 |
230 |
|||||||
818 |
162 |
411 |
264 |
203 |
|||||||
2AA 2-Aminoanthracene
BP Benzo(a)pyrene
P Precipitate
# Standard deviation
Tables 4 and 5 below
Table: Summary of the Mouse Lymphoma Assay on Aluminum, benzoate C16-18-fatty acids complexes (41205678)
Experiment 1
Treatment (µg/ml) |
4-Hours-S9 |
Treatment (µg/ml) |
4-Hours+S9 |
||||||||||
|
%RSG |
RTG |
MF§ |
|
%RSG |
RTG |
MF§ |
||||||
0 |
|
100 |
1.00 |
123.96 |
|
0 |
|
100 |
1.00 |
128.09 |
|
||
4.88 |
Ø |
91 |
|
|
|
39.06 |
Ø |
107 |
|
|
|
||
9.77 |
Ø |
92 |
|
|
|
78.13 |
Ø |
100 |
|
|
|
||
19.53 |
|
90 |
0.83 |
129.32 |
|
156.25 |
|
89 |
0.75 |
141.53 |
|
||
39.06 |
|
84 |
0.91 |
134.73 |
|
312.5 |
|
69 |
0.51 |
120.70 |
|
||
78.13 |
|
81 |
0.72 |
116.77 |
|
468.75 |
|
64 |
0.44 |
136.24 |
|
||
156.25 |
|
64 |
0.61 |
143.60 |
|
625 |
|
46 |
0.29 |
146.90 |
|
||
312.5 |
|
42 |
0.37 |
153.18 |
|
937.5 |
|
20 |
0.08 |
196.66 |
* |
||
468.75 |
|
21 |
0.12 |
160.99 |
|
1250 |
X |
13 |
0.02 |
369.05 |
|
||
Linear trend |
|
* |
Linear trend |
|
* |
||||||||
EMS |
|
|
|
|
|
CP |
|
|
|
|
|
||
400 |
|
60 |
0.46 |
1022.04 |
|
2 |
|
55 |
0.24 |
1459.85 |
|
||
|
|
|
|
|
|
|
|
|
|
|
|
||
Experiment 2
Treatment (µg/ml) |
24-Hours-S9 |
Treatment (µg/ml) |
4-Hours+S9 |
||||||||
|
%RSG |
RTG |
MF§ |
|
%RSG |
RTG |
MF§ |
||||
0 |
|
100 |
1.00 |
108.39 |
|
0 |
|
100 |
1.00 |
110.11 |
|
5 |
Ø |
95 |
|
|
|
40 |
Ø |
96 |
|
|
|
10 |
|
96 |
0.77 |
103.57 |
|
80 |
|
90 |
0.97 |
119.73 |
|
20 |
|
73 |
0.78 |
105.22 |
|
160 |
|
70 |
0.82 |
116.68 |
|
40 |
|
46 |
0.58 |
127.28 |
|
320 |
|
38 |
0.30 |
143.02 |
|
60 |
|
42 |
0.58 |
105.28 |
|
480 |
|
37 |
0.30 |
128.14 |
|
80 |
|
16 |
0.26 |
126.27 |
|
640 |
|
29 |
0.22 |
123.00 |
|
120 |
|
10 |
0.16 |
128.14 |
|
800 |
X |
19 |
0.05 |
298.62 |
|
160 |
Ø |
2 |
|
|
|
960 |
X |
13 |
0.02 |
293.81 |
|
Linear trend |
|
NS |
Linear trend |
|
NS |
||||||
EMS |
|
|
|
|
|
CP |
|
|
|
|
|
150 |
|
38 |
0.33 |
1112.59 |
|
2 |
|
55 |
0.30 |
767.84 |
|
MF§ 5 -TFT resistant mutants/106viable cells 2 days after treatment
% Percent relative survival adjusted by post treatment cell count factor
RTG Relative total growth adjusted to account for immediate post-treatment toxicity
Ø Not plated for viability / 5-TFT resistance due to toxicity or surplus to requirements
X Treatment excluded from test statistics due to toxicity
* Significant at 5%
NS Not significant
Table 2. CBPI – Experiment 1
4-Hour exposure without S9 |
4-Hour exposure without S9 (2%) |
||||||||||||||
Dose level (µg/ml) |
Replicate |
Nucleate cells/500 cells |
CBPI |
Mean CBPI |
% control CBPI |
Dose level (µg/ml) |
Replicate |
Nucleate cells/500 cells |
CBPI |
Mean CBPI |
% control CBPI |
||||
Mono |
Bi |
Multi |
Mono |
Bi |
Multi |
||||||||||
0 |
A |
190 |
230 |
80 |
1.78 |
1.58 |
0 |
0 |
A |
126 |
254 |
120 |
1.99 |
1.96 |
100 |
B |
344 |
118 |
38 |
1.39 |
B |
130 |
275 |
95 |
1.93 |
||||||
75 |
A |
167 |
231 |
102 |
1.87 |
1.66 |
1.12 |
75 |
A |
264 |
180 |
56 |
1.58 |
1.79 |
82 |
B |
311 |
158 |
31 |
1.44 |
B |
118 |
266 |
116 |
2.00 |
||||||
150 |
A |
168 |
261 |
71 |
1.81 |
1.73 |
125 |
150* |
A |
327 |
150 |
23 |
1.39 |
1.68 |
71 |
B |
219 |
234 |
47 |
1.68 |
B |
132 |
252 |
116 |
1.97 |
||||||
300† |
A |
219 |
240 |
41 |
1.64 |
1.58 |
99 |
225* |
A |
209 |
253 |
38 |
1.66 |
1.80 |
83 |
B |
261 |
219 |
20 |
1.52 |
B |
122 |
287 |
91 |
1.94 |
||||||
600P† |
A |
NSB |
NSB |
NSB |
NSB. |
NSB |
NSB |
300P* |
A |
194 |
226 |
80 |
1.77 |
1.79 |
82 |
B |
NSB |
NSB |
NSB |
NSB. |
B |
162 |
276 |
62 |
1.80 |
||||||
900P† |
A |
NB |
NB |
NB |
NB |
NB |
NB |
450P* |
NSB |
NSB |
NSB |
NSB. |
NSB |
NSB |
NSB |
B |
NB |
NB |
NB |
NB |
NSB |
NSB |
NSB |
NSB. |
NSB |
||||||
1200P† |
A |
NB |
NB |
NB |
NB |
NB |
NB |
600P* |
NSB |
NSB |
NSB |
NSB. |
NSB |
NSB |
NSB |
B |
NB |
NB |
NB |
NB |
NSB |
NSB |
NSB |
NSB. |
NSB |
||||||
MMC 0.2 |
A |
244 |
239 |
17 |
1.55 |
1.54 |
92 |
CP 5 |
A |
260 |
234 |
6 |
1.49 |
1.48 |
50 |
B |
248 |
237 |
15 |
1.53 |
B |
274 |
219 |
7 |
1.47 |
||||||
MMC = Mitomycin C
CP = Cyclophosphamide
NB = No binucleate cells
NSB = No binucleate cells suitable for scoring
P = Precipitate observed at the end of exposure period
† = Precipitate observed on the slides
Table 3: CBPI – Experiment 2
20-Hour exposure without S9 |
|||||||
Dose level (µg/ml) |
Replicate |
Nucleate cells/500 cells |
CBPI |
Mean CBPI |
% Control CBPI |
||
Mono |
Bi |
Multi |
|||||
0 |
A |
48 |
326 |
126 |
2.16 |
2.20 |
100 |
B |
41 |
300 |
159 |
2.25 |
|||
75P |
A |
52 |
328 |
120 |
2.14 |
2.07 |
89 |
B |
87 |
325 |
88 |
2.00 |
|||
150P |
A |
83 |
314 |
103 |
2.04 |
2.11 |
93 |
B |
41 |
325 |
134 |
2.19 |
|||
300P† |
A |
129 |
299 |
72 |
1.89 |
1.87 |
73 |
B |
158 |
259 |
83 |
1.85 |
|||
600P† |
A |
NSB |
NSB |
NSB |
NSB |
NSB |
NSB |
B |
NSB |
NSB |
NSB |
NSB |
|||
900P† |
A |
NSB |
NSB |
NSB |
NSB |
NSB |
NSB |
B |
NSB |
NSB |
NSB |
NSB |
|||
1200P† |
A |
NSB |
NSB |
NSB |
NSB |
NSB |
NSB |
B |
NSB |
NSB |
NSB |
NSB |
|||
DC 0.075 |
A |
251 |
206 |
43 |
1.58 |
1.58 |
48 |
B |
249 |
214 |
37 |
1.58 |
|||
DC = Demecolcine
NSB = No binucleate cells suitable for scoring
P = Precipitate observed at the end of exposure period
† = Precipitate observed on the slides
.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
The assessment of genetic toxicity of the registered substance is based upon read-across data from key studies performed on aluminum, benzoate C16 -18 fatty acid complexes. For the genetic toxicity of the substances, read across from the source to the target substance is considered justified. Both substances would not leach when in situ in base oil during use as grease thickeners and are not expected to be bioavailable. The substances would dissociate into inorganic aluminium species and fatty acids (plus benzoic acid for the source substance), the organic components of which are readily metabolised. As the fatty acid components are essential nutrients to many organisms and are not expected to be hazardous (and the benzoate component of the source substance is not expected to be hazardous), the toxicity is expected to be driven by the aluminium component, so would be the same in both the source and target substances. As such, read across from the source substance is considered to provide a worst-case scenario for the target substance.
A key bacterial reversion assay (Ames test) was conducted on aluminum, benzoate C16-18-fatty acids complexes together with a micronucleus assay in human lymphocytes. In the Ames test, no significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria at any dose level, up to the maximum recommended, either with or without metabolic activation or exposure method. In the micronucleus test, aluminum, benzoate C16-18-fatty acids complexes did not induce statistically significant increase in the frequency of cells with micronuclei in cultured human peripheral blood lymphocytes when tested up to a limit of solubility in both the absence and presence of a rat liver metabolic activation system (S-9). In the case of both studies, the results were negative, therefore aluminum, benzoate C16-18-fatty acids complexes was considered to be non-mutagenic, non-clastogenic and non-aneugenic under the conditions of these tests. As a consequence of the negative results in both key assays, a second mutation test was performed in accordance with Annex VII REACH regulations in a mammalian cell system namely, the mouse lymphoma assay. This test was also negative.
Justification for classification or non-classification
Classification for mutagenicity generally requires positive results from appropriate in vivo studies. Negative results from the three key read-across in vitro studies with aluminum, benzoate C16-18 fatty acid complexes would suggest that testing in vivo for the registered substance is not necessary. Therefore, the substance is not classified for genetic toxicity as a consequence of negative results in all studies conducted on the read-across substance, aluminum, benzoate C16-18 fatty acid complexes.
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.
