Methacrylaldehyde

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:

2018

Report date:

2018

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:

Methacrylaldehyde

EC Number:

201-150-1

EC Name:

Methacrylaldehyde

Cas Number:

78-85-3

Molecular formula:

C4H6O

IUPAC Name:

methacrylaldehyde

Test material form:

liquid

Method

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:

S9 mix from male Wistar rats livers

Test concentrations with justification for top dose:

1st Experiment: 0; 33; 100; 333; 1000; 2600 and 5200 μg/plate (without S9 mix, SPT)
2nd Experiment: 0; 10; 33; 100; 333; 1000; 2600 and 5200 μg/plate (with S9 mix, SPT); 0; 10; 33; 100; 333; 1000; and 2600 μg/plate (TA 1537 and TA 98 without S9 mix, SPT)
3rd Experiment: 0; 333; 500; 800; 1000; 2000; 3500 and 5200 μg/plate (TA 1535 with S9 mix, SPT) 0; 1000; 1500; 2600; 3500 and 5200 μg/plate (TA 100 with S9 mix, SPT)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility of the test substance in water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

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:

Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al. (1, 2).
Salmonella typhimurium
Due to heat sensitivity of the test substance, the experiment was performed without water bath. The following components were added to the test tubes:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
Subsequently, 2 mL 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) was added and, after mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°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 (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
Escherichia coli
Due to heat sensitivity of the test substance, the experiment was performed without water bath. The following components were added to the test tubes:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
Subsequently, 2 mL 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) was added and, after mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°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 (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Evaluation 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.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from about 2600 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from about 2600 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

other: TA 1537, TA 98, E.coli WP2 uvrA

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from about 2600 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

other: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from about 2600 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A bacteriotoxic effect was observed depending on the strain and the test conditions from about 2600 μg/plate onward.

MUTAGENICITY: A biologically relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100 and TA 98 or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test using tester strains TA 1537, TA 98 and E. coli WP2 uvrA with and without S9 mix. Furthermore, using tester strains TA 1535 and TA 100 without metabolic activation no relevant increase in the number of his+ revertants was observed in standard plate test.

A relevant, reproducible and partly dose dependent increase in the number of his+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed with TA 100 with metabolic activation.

TA 100 (with S9 mix): Increase at concentrations of 2600 and 5200 μg/plate in the 2nd Experiment and at concentrations of 1500, 2600, 3500 and 5200 μg/plate in the 3rd Experiment.

Using tester strains TA 1535, a relevant increase of his+ revertants exceeding a factor of 3 compared to the concurrent vehicle control was observed with metabolic activation.

TA 1535 (with S9 mix): Increase at concentrations of 1000, 2000 and 3500 μg/plate in the 3rd Experiment.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance Methacrolein is mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the presence of metabolic activation.