Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.

REACH

[No public or meaningful name is available]

EC number: - | CAS number: -

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (Ames test, OECD 471): negative with and without metabolic activation

Link to relevant study records

Reference

Endpoint:: in vitro gene mutation study in bacteria
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 28 Oct - 16 Nov 2020
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:: adopted in 2020
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:: adopted in 2008
Deviations:: no
Qualifier:: according to guideline
Guideline:: EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:: adopted in 1998
Deviations:: no
Qualifier:: according to guideline
Guideline:: other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
Deviations:: no
Qualifier:: according to guideline
Guideline:: other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register)
Version / remarks:: Adopted in 2012; 77:33748-33749
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Type of assay:: bacterial reverse mutation assay
Target gene:: his operon (for S. typhimurium strains) and trp operon (for E. coli strain)
Species / strain / cell type:: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):: CELLS USED
- Source of cells and date obtained: British Industrial Biological Research Association, obtained 17 August 1987, and Trinova Biochem GmbH obtained 27 June 2017

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Top agar was prepared using 0.6% Bacto agar (lot number 9294156 expiry date 08/2024) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 56249 expiry date 11/2020 and 56310 expiry date 12/2020).
Species / strain / cell type:: E. coli WP2 uvr A
Details on mammalian cell type (if applicable):: CELLS USED
- Source of cells and date obtained:
British Industrial Biological Research Association, obtained 17 August 1987, and Trinova Biochem GmbH obtained 27 June 2017

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Top agar was prepared using 0.6% Bacto agar (lot number 9294156 expiry date 08/2024) and 0.5% sodium chloride with 5 mL of 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 56249 expiry date 11/2020 and 56310 expiry date 12/2020).
Metabolic activation:: with and without
Metabolic activation system:: Type and composition of metabolic activation system: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague-Dawley rats (male, 5 - 6 weeks, 175 - 199 g) treated with phenobarbitone / β-naphthoflavone
- source of S9 : purchased from Moltox/Trinova Biochem GmbH, Giessen, Germany (Lot No. 4222)
- Protein level in S9: 20 mg/mL
- Method of preparation of S9 mix: S9-mix was prepared before use using sterilised co-factors and maintained on ice for the duration of the test
- Composition of S9 mix:
S9: 5.0 mL
1.65 M KCl/0.4 M MgCl2: 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL
- Concentration or volume of S9 mix in the final culture medium: 0.5 mL (10%)
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): checked at producer and specified in quality control certificate
Test concentrations with justification for top dose:: Experiment 1 (plate incorporation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (5000 μg/plate being the maximum recommended dose level)
Experiment 2 (pre-incubation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (determined by the results of Experiment 1)
Vehicle / solvent:: - Vehicle(s)/solvent(s) used: acetone
- Purity: > 99.75%
- Supplier: Acros Organics
- Batch number: 1911510
- Expiry: Nov 2021

- Justification for choice of solvent/vehicle: the test substance was not soluble/miscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully miscible in acetone at 100 mg/mL

- Justification for percentage of solvent in the final culture medium: acetone is toxic to the bacterial cells at 0.1 mL (100 μL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates; to compensate, each formulation was dosed using 0.05 mL (50 μL) aliquots.
Untreated negative controls:: yes
Negative solvent / vehicle controls:: yes
True negative controls:: no
Positive controls:: yes
Positive control substance:: 4-nitroquinoline-N-oxide; 9-aminoacridine; N-ethyl-N-nitro-N-nitrosoguanidine; benzo(a)pyrene; other: 2-Aminoanthracene
Details on test system and experimental conditions:: NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate
- Number of independent experiments: two independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation, Experiment 1) and preincubation (Experiment 2)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min at 37 ± 3 °C (Experiment 2)
- Exposure duration/duration of treatment: 48 - 72 h at 37 ± 3 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn

METHODS FOR MEASUREMENT OF GENOTOXICIY
- Method: count of revertant colonies
Evaluation criteria:: Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response.
5. Statistical analysis of data as determined by UKEMS.
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:: Mean values and standard deviation were calculated. Statistical significance was confirmed using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies.
: Key result
Species / strain:: S. typhimurium TA 1535
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Experiment 1 (plate incorporation): 1500 μg/plate +/- S9-mix; Experiment 2 (pre-incubation): 1500 µg/plate -S9, 5000 µg/plate +S9
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not examined
Positive controls validity:: valid
: Key result
Species / strain:: S. typhimurium TA 1537
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Experiment 1 (plate incorporation): 1500 μg/plate +/- S9-mix; Experiment 2 (pre-incubation): 500 µg/plate -S9, 1500 µg/plate +S9
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not examined
Positive controls validity:: valid
: Key result
Species / strain:: S. typhimurium TA 98
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Experiment 1 (plate incorporation): 1500 μg/plate +/- S9-mix; Experiment 2 (pre-incubation): 500 µg/plate -S9, 1500 µg/plate +S9
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not examined
Positive controls validity:: valid
: Key result
Species / strain:: S. typhimurium TA 100
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Experiment 1 (plate incorporation): 1500 μg/plate +/- S9-mix; Experiment 2 (pre-incubation): 500 µg/plate -S9, 1500 µg/plate +S9
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not examined
Positive controls validity:: valid
: Key result
Species / strain:: E. coli WP2 uvr A
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Experiment 1 (plate incorporation): 1500 μg/plate +/- S9-mix; Experiment 2 (pre-incubation): 1500 µg/plate -S9, 5000 µg/plate +S9
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not examined
Positive controls validity:: valid
Additional information on results:: TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: A test item precipitate (globular in appearance) was noted at and above 1500 μg/plate in both the presence and absence of metabolic activation (S9-mix) in both experiments. This observation did not prevent the scoring of revertant colonies.

STUDY RESULTS
- Concurrent vehicle negative and positive control data provided as pdf file under 'Attached background material'.
- Study results fall within the negative historical control data range.

Ames test:
- Detailed results are provided in tabular form under 'Any other information on results incl. tables'.
Conclusions:: Interpretation of results: negative

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:: no study available

Additional information

Gene mutation in bacteria (Ames test)

The potential of fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics (EC No. 942-654-8) to induce gene mutation in bacteria was assessed in a reverse mutation assay according to OECD guideline 471 under GLP conditions (Covance, 2021d). Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the plate incorporation and pre-incubation methods at eight 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 for Experiment 1 (plate incorporation) was based on the recommendation given in the test guideline and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was the same as in Experiment 1 (1.5 to 5000 µg/plate). Eight test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least five analysable dose levels as required by the test guideline, and were selected based on the cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation. The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item induced a visible reduction in the growth of the bacterial background lawn of all of the tester strains at and above 1500 µg/plate in both the presence and absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). The test item induced a visible reduction in the growth of the bacterial background lawn of all of the tester strains dosed in the absence of metabolic activation (S9-mix) from 500 µg/plate to TA100, TA98 and TA1537 and 1500 µg/plate to TA1535 and WP2uvrA. In the presence of metabolic activation (S9-mix), toxicity was noted from 1500 µg/plate to TA100, TA98 and TA1537 and at 5000 µg/plate to TA1535 and WP2uvrA. A test item precipitate (globular in appearance) was noted at and above 1500 µg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This observation did not prevent the scoring of revertant colonies. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). A small, statistically significant increase in TA1537 revertant colony frequency was observed in the presence of S9-mix at 15 µg/plate. This increase was considered to be of no biological relevance because there was no evidence of a dose response relationship and the fold increase was only 1.5 times the concurrent vehicle control. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). In this reverse mutation assay (Ames test) fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics (EC No. 942-654-8) did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test the substance was considered to be non-mutagenic.

Justification for classification or non-classification

The available data on in vitro genetic toxicity of fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics (EC No. 942-654-8) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
162		34		13		20		12
149	(153)	36	(34)	26	(21)	17	(19)	12	(11)
148		32		25		20		9
Viability – Bacterial cells 10⁹ per mL
2.6		2.4		2.5		1.4		2.1