Registration Dossier

Diss Factsheets

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:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
This study was performed to investigate the potential of Macrolex Fluoreszenzrot 4B to induce gene mutations according to the plate incorporation test (experiment I and Ia) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA, following OECD TG 471.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione
EC Number:
244-320-0
EC Name:
6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione
Cas Number:
21295-57-8
Molecular formula:
C23H22N2O2
IUPAC Name:
10-(cyclohexylamino)-14-methyl-14-azatetracyclo[7.7.1.0²,⁷.0¹³,¹⁷]heptadeca-1(16),2,4,6,9,11,13(17)-heptaene-8,15-dione
Test material form:
solid
Details on test material:
Purity: 94.2%

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
Test concentrations with justification for top dose:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate.
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate.
Vehicle / solvent:
On the day of the experiment, the test item Macrolex Fluoreszenzrot 4B was suspended in DMSO (purity > 99%). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD; 2-aminoanthracene, 2-AA
Details on test system and experimental conditions:
This study was performed to investigate the potential of Macrolex Fluoreszenzrot 4B to induce gene mutations according to the plate incorporation test (experiment I and Ia) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA, following OECD TG 471.
The assay was performed in two independent experiments. Experiment I was performed with and without liver microsomal activation using all 5 strains, whereas experiment Ia was performed with liver microsomal activation only and solely using TA 1537. Each concentration, including the controls, was tested in triplicate. The test item was tested as solution in DMSO at the following concentrations in both experiments:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Rationale for test conditions:
Pre-Experiment for Toxicity:
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I since the acceptance criteria are met.

Dose Selection
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since a positive result was obtained in the first experiment, a second experiment was not performed.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Key result
Species / strain:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
TA 1537 was positive in the presence of S9 mix.
Cytotoxicity / choice of top concentrations:
other: No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
During the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 1537 in the presence of S9 mix.

Applicant's summary and conclusion

Conclusions:
Macrolex Fluoreszenzrot 4B is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of Macrolex Fluoreszenzrot 4B to induce gene mutations according to the plate incorporation test (experiment I and Ia) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA, following OECD TG 471.


The assay was performed in two independent experiments. Experiment I was performed with and without liver microsomal activation using all 5 strains, whereas experiment Ia was performed with liver microsomal activation only and solely using TA 1537. Each concentration, including the controls, was tested in triplicate. The test item was tested as solution in DMSO at the following concentrations in both experiments:


3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate


The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate. The test item partially adhered in the test tubes from 333 μg/plate onwards in the presence of S9 mix. No precipitation of the test item, but a strong coloring of the overlay agar on the incubated agar plates (densely colored plates) was observed from 2500 to 5000 μg/plate.


The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.


No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.


A moderate but dose dependent increase in revertant colony numbers was observed following treatment with Macrolex Fluoreszenzrot 4B in strain TA 1537 in the presence of metabolic activation (S9 mix). The threshold of three times the revertant colony number of the corresponding solvent control was exceeded at only one concentration (1000 μg/plate). To validate the positive response strain TA 1537 with S9 mix was repeated under the same conditions as experiment I. The repeated experiment is reported as experiment Ia. Also in the repeated experiment an increase in revertant colony count was observed. The threshold of threefold the number in revertant colony count of the corresponding solvent control was exceeded at 5000 μg/plate. Therefore the positive effect observed in experiment I is considered as reproducible and the test item Macrolex Fluoreszenzrot 4B as mutagenic in this Ames test.


Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.


Conclusion


In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 1537 in the presence of S9 mix.


Therefore, Macrolex Fluoreszenzrot 4B is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay