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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)

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)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3,4-dihydrocoumarin
EC Number:
204-354-9
EC Name:
3,4-dihydrocoumarin
Cas Number:
119-84-6
Molecular formula:
C9H8O2
IUPAC Name:
chroman-2-one

Method

Target gene:
Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation.
Vehicle / solvent:
DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this sol-vent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
mitomycin C
other: 4-Nitro-1,2-pheny-lene diamine, Benzo-a-pyrene, 2-Amino-anthracene
Details on test system and experimental conditions:
Per bacteria strain and concentration, three plates with (+S9) and three plates without met-abolic activation (-S9) were used.
The test item solutions were prepared according to chapter 6.1.4, page 12.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ± 1 °C.

Plate incorporation method:
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
1. 100 μL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control). For the positive control MMC 2.5 μL of the stock solu-tion were applied to achieve a final concentration of 0.5 μL/plate.
2. 500 μL S9-mix (see chapter 6.4.13, page 18 for test with metabolic activation) or phos-phate buffer (for test without metabolic activation).
3. 100 μL bacteria suspension (see chapter 6.2.2, page 13, test system, culture of the strains)
4. 2000 μL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ± 1 °C.

Pre-incubation method:
The following materials were gently vortexed in a test tube and incubated at 37 ± 1 °C for 20 minutes:
1. 100 μL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control). For the positive control MMC 2.5 μL of the stock solu-tion were applied to achieve a final concentration of 0.5 μL/plate.
2. 500 μL S9-mix (see chapter 6.4.13, page 18 for test with metabolic activation) or phos-phate buffer (for test without metabolic activation).
3. 100 μL bacteria suspension (see chapter 6.2.2, page 13, test system, culture of the strains)
After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently slewed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ± 1 °C.
Evaluation criteria:
Five different analysable and non-toxic concentrations were used for the evaluation of the mutagenic potential of the test item.
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, negative control and positive control.
The mean values and standard deviations of each threefold determination were calculated as well as the increase factor of revertant induction (mean revertants divided by mean spon-taneous revertants) of the test item solutions and the positive controls. Additionally, the ab-solute number of revertants (mean revertants minus mean spontaneous revertants) is given.
A result is considered as positive if a clear and dose-related increase in the number of re-vertants occurs and/or a biologically relevant positive response for at least one of the con-centrations occurs in at least one tested strain with or without metabolic activation.

A biologically relevant increase is described as follows:
1. if in the bacteria strains S. typhimurium TA98, TA100, TA102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase factor of at least 2.0)
2. if in the bacteria strains S. typhimurium TA1535 and TA1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3.0).

A substance is not mutagenic if it does not meet the criteria above.
If the criteria listed above are not clearly met, the results will be assessed as equivocal and will be discussed.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
3,4-dihydrocoumarin is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

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