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Genetic toxicity in vitro

Description of key information

The in vitro genotoxicity of 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) was investigated using a combination of valid testing and non-testing methods.

 

The capacity of 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) to induce gene mutation in bacteria was evaluated during a GLP-compliant study performed in accordance with the OECD Testing Guideline 471. Bacterial strains were Salmonella typhimurium TA1537, TA98, TA1535 and TA100, and Escherichia coli WP2uvrA. Experiments were performed with and without metabolic activation using S9-mix. Acetone was selected as a vehicle and used for negative control, giving counts of revertant colonies within the normal range. Relevant positive controls were selected for each bacteria strains in accordance with the OECD Testing Guideline 471 and induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Therefore all the controls were considered as valid. Plate incorporation method (experiment 1) and pre-incubation method (experiment 2) were used. The maximum dose level of the test item was 5,000 μg/plate in both experiments. In the first experiment (plate incorporation method), the maximum dose level of the test item was 5000 μg/plate (the maximum concentration). The test item induced toxicity as weakened bacterial background lawns at 5000 μg/plate for one strain of Salmonella only (TA100 dosed in the absence of S9-mix). No visible reduction in the growth of the bacterial background lawns were noted for any of the remaining tester strains.

In experiment 2 there was no visible reduction in the growth of the bacterial background lawn noted to any of the tester strains at any dose level, either in the presence or absence of metabolic activation (S9-mix). There were no toxicologically meaningful 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). Small, statistically significant increases in revertant colony frequency were observed in Experiment 1 (absence of S9-mix) at 15 μg/plate (TA100 and TA1535) and in the presence of S9-mix at 15, 150 and 500 μg/plate (TA100). However, these responses were within the in-house historical vehicle/untreated control values for the bacterial strains and were, therefore, considered of no biological relevance. Similarly, no 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). It is therefore concluded that 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) was not mutagenic under the conditions of the test.

 

Assessments were performed based on QSAR predictions, concluding that 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) would not induce structural chromosomal aberration nor gene mutation in mammalian cells.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
other: expert assessment
Adequacy of study:
weight of evidence
Study period:
2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: An assessment was performed based on QSAR predictions
Qualifier:
no guideline required
Principles of method if other than guideline:
An assessment was performed based on QSAR predictions
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Key result
Species / strain:
other: not applicable
Metabolic activation:
not applicable
Genotoxicity:
other: not applicable
Cytotoxicity / choice of top concentrations:
other: not applicable
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: An assessment was performed based on QSAR predictions
Conclusions:
2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) does not induce structural chromosomal aberration in mammalian cells.
Executive summary:

An assessment was performed based on QSAR predictions. It concluded that 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) is unlikely to induce structural chromosomal aberration in mammalian cells.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 01 December 2017 to 12 January 2018
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:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus in the genome of Salmonella typhimurium and tryptophan locus in the genome of Escherichia coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Experiment 1: 1.5, 5, 15, 50, 150, 500, 1,500 and 5,000 μg/plate
Experiment 2: 5, 15, 50, 150, 500, 1,500, 5,000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
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
not specified
Details on test system and experimental conditions:
The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in acetone by mixing on a vortex mixer. The test item was confirmed as a UVCB product and, therefore, no correction for purity was required. 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 (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
The test item was tested using the following method. The maximum concentration was 5,000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1,500 and 5,000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added together with 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer to 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
With metabolic activation, the procedure was the same except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
All of the plates were incubated at 37 ± 3°C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 5, 15, 50, 150, 500, 1,500, 5,000 μg/plate.
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the test item formulation or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3°C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
Seven test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
With metabolic activation, the procedure was the same except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3°C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Rationale for test conditions:
In accordance with the OECD Testing Guideline 471.
Evaluation criteria:
There are several criteria for determining a positive result. 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 (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item 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 making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
There was no dose-related statistically significant increase in number of revertants outside historical data in both experiments. It was concluded that 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) was not mutagenic under the conditions of the test.
Executive summary:

The capacity of 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) to induce gene mutation in bacteria was evaluated during a GLP-compliant study performed in accordance with the OECD Testing Guideline 471.

Bacterial strains were Salmonella typhimurium TA1537, TA98, TA1535 and TA100, and Escherichia coli WP2uvrA. Experiments were performed with and without metabolic activation using S9-mix.

Acetone was selected as a vehicle and used for negative control, giving counts of revertant colonies within the normal range. Relevant positive controls were selected for each bacteria strains in accordance with the OECD Testing Guideline 471 and induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Therefore all the controls were considered as valid.

Plate incorporation method (experiment 1) and pre-incubation method (experiment 2) were used. The maximum dose level of the test item was 5,000 μg/plate in both experiments.

In the first experiment (plate incorporation method), the maximum dose level of the test item was 5000 μg/plate (the maximum concentration). The test item induced toxicity as weakened bacterial background lawns at 5000 μg/plate for one strain of Salmonella only (TA100 dosed in the absence of S9-mix). No visible reduction in the growth of the bacterial background lawns were noted for any of the remaining tester strains.

In experiment 2there was no visible reduction in the growth of the bacterial background lawn noted to any of the tester strains at any dose level, either in the presence or absence of metabolic activation (S9-mix).

There were no toxicologically meaningful 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). Small, statistically significant increases in revertant colony frequency were observed in Experiment 1 (absence of S9-mix) at 15 μg/plate (TA100 and TA1535) and in the presence of S9-mix at 15, 150 and 500 μg/plate (TA100). However, these responses were within the in-house historical vehicle/untreated control values for the bacterial strains and were, therefore, considered of no biological relevance.

Similarly, no 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).

It is therefore concluded that 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) was not mutagenic under the conditions of the test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
other: expert assessment
Adequacy of study:
weight of evidence
Study period:
2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: An assessment was performed based on QSAR predictions
Qualifier:
no guideline required
Principles of method if other than guideline:
An assessment was performed based on QSAR predictions
GLP compliance:
no
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Key result
Species / strain:
other: not applicable
Metabolic activation:
not applicable
Genotoxicity:
other: not applicable
Cytotoxicity / choice of top concentrations:
other: not applicable
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: An assessment was performed based on QSAR predictions
Conclusions:
2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) does not induce gene mutation in mammalian cells.
Executive summary:

An assessment was performed based on QSAR predictions. It concluded that 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) is unlikely to induce gene mutation in mammalian cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The capacity of 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) to induce gene mutation in bacteria and mammalian cells and structural chromosomal aberration in mammalian cells was investigated by performing an in vitro study according to a relevant OECD Testing Guideline and expert assessments based on valid QSAR predictions. Results from the study and expert assessment were negative.

 

In accordance with Annexes VII and VIII of REACH it was not required to consider in vivo genotoxicity testing on 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) as no positive result was obtained.

 

As a conclusion 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) does not meet the criteria for classification as mutagenic in accordance with Regulation (EC) N°1272/2008.