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Toxicological information

Genetic toxicity: in vitro

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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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Species / strain
Species / strain:
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:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility of the test substance in water
Controls
Negative controls:
yes
Solvent 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 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 resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
from about 2600 μg/plate onward
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
from about 2600 μg/plate onward
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
other: TA 1537, TA 98, E.coli WP2 uvrA
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
from about 2600 μg/plate onward
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
other: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
from about 2600 μg/plate onward
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

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.