2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters

EC Number:

292-124-9

EC Name:

2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters

Cas Number:

90552-04-8

Molecular formula:

C(17-19)H(32-36)O2

IUPAC Name:

2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters

Test material form:

liquid

Method

Target gene:

his, trp

Species / strain

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:

Type and composition of metabolic activation system:
S9 mix from male Wistar rats liver; induced with phenobarbital and ß-napthoflavone

Test concentrations with justification for top dose:

1st Exp: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (with and without S9 mix; SPT)
2nd Exp: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (with and without S9 mix, PIT)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ultrapure water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, water was used as vehicle.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine; 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : 3 test plates per dose or per control

Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al.
Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order: 0.1 mL test solution, vehicle or positive control; 0.1 mL fresh bacterial culture;0.5 mL S9 mix (with external metabolic activation) or 0.5 mL phosphate buffer (without external metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 seconds. After incubation at 37°C (±2°C) for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager. Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order: 0.1 mL test solution, vehicle or positive control; 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with external metabolic activation) or 0.5 mL phosphate buffer (without external metabolic activation).
After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 seconds. After incubation at 37°C (±2°C) for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager. Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al.
0.1 mL test solution, vehicle or positive control, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with external metabolic activation) or phosphate buffer (without external metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds. After incubation at 37°C (±2°C) for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager. Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System. Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Evaluation criteria:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain
The sterility controls revealed no indication of bacterial contamination.
The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies compatible with the range of the historical positive control data or above.
Fresh bacterial culture containing approximately 109 cells per mL were used.
Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix
or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

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

Positive controls validity:

valid

Additional information on results:

SOLUBILITY: Precipitation of the test substance was observed at and above 1000 μg/plate with and without S9 mix.
TOXICITY: A bacteriotoxic effect only was observed in the preincubation test depending on the strain and test conditions at and above 2500 μg/plate
MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (increased by a factor of 2 or above compared to the concurrent control for Salmonella typhimurium TA 100, TA 98 and Escherichia coli WP2 uvrA, or a factor of 3 or above for Salmonella typhimurium TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test with or without the addition of an external metabolizing system (liver S9 mix of rats treated with enzyme inducers).

Any other information on results incl. tables

Toxicity: Decreased revertant numbers were observed at following concentrations (μg/plate):

Experiment	S9	TA1535	TA 100	TA 1537	TA 98	E.coli
1st-SPT	without	-	-	-	-	-
	with	-	-	-	-	-
2nd-PIT	without	-	-	-	5000	-
	with	5000	-	≥2500	≥2500	-

- = no adverse effect observed

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, it is concluded that 2-Propenoic acid, 2- methyl-, C13-15-branched and linear alkyl esters is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of external metabolic activation.