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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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:
ethyl (2S)-3-[3,5-diamino-4-(4-methoxyphenoxy)phenyl]-2-acetamidopropanoate
Cas Number:
440667-78-7
Molecular formula:
C20H25N3O5
IUPAC Name:
ethyl (2S)-3-[3,5-diamino-4-(4-methoxyphenoxy)phenyl]-2-acetamidopropanoate
Test material form:
solid: particulate/powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Test concentrations with justification for top dose:
The following nominal concentrations were prepared for experiment 1 and 1b:
5000 µg/plate, 1500 µg/plate, 500 µg/plate, 150 µg/plate and 50 µg/plate.

The following nominal concentrations were prepared for the second experiment:
5000 µg/plate, 2500 µg/plate, 1250 µg/plate, 625 µg/plate, 313 µg/plate and 156 µg/plate.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Remarks:
DMSO and water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO and water
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-Amino-anthracene, C14H11N; CAS-No.: 613-13-8 4-Nitro-1,2-phenylene diamine, C6H7N3O2; CAS-No.: 99-56-9
Details on test system and experimental conditions:
7.1 Culture of Bacteria
On the day before the start of each experiment, a nutrient broth (Oxoid nutrient broth no. 2) was inoculated with one lyophilizate per strain at 4:00 pm.
These overnight cultures were placed in the heating chamber at 37 ± 1 °C for 16 hours.
For the last two hours the overnight cultures were shaken on an orbital shaker (150 rpm) at 37 ± 1 °C.
Afterwards, the overnight cultures were ready for usage in the experiment.
During the test, the overnight cultures were stored at room temperature (20 ± 5 °C) as to prevent changes in the titre.
7.2 Conduct of Experiment
7.2.1 Preparations
Different media and solutions were prepared preliminary (exact production dates are docu-mented in the raw data).
On the day of the test, the bacteria cultures were checked for growth visually. The incuba-tion chambers were heated to 37 ± 1 °C. The water bath was turned to 43 ± 1 °C. The table surface was disinfected.
The S9 mix was freshly prepared and stored at 0 °C.

Results and discussion

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

Applicant's summary and conclusion

Conclusions:
The test item L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester showed no increase in the number of revertants in all bacteria strains in both experiments.
All negative and nearly all strain-specific positive control values were within the laboratory historical control data ranges (see chapter 17, page 49) indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester is not mutagenic in the Salmonella typhimurium test strains TA98, TA100, TA102, TA1535 and TA1537 in the absence and presence of metabolic acti-vation under the experimental conditions in the present study.
In all experiments, the test item caused no cytotoxicity towards all bacteria strains.
The confirmation tests of the genotype performed by TRINOVA BioChem GmbH did not show any irregularities. The control of the titre was above the demanded value of 109 bac-teria/mL.
All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. Nearly all numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory (see chapter 17, page 49). All numbers of revertant colonies of the positive controls were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.
Since all criteria for acceptability have been met, the study is considered valid.
Executive summary:

Findings and Results:


Three experiments were performed.


Experiment 1 was invalid for TA1537 and had to be repeated due to an experimental error.


 


The study procedures described in this report were based on the most recent Guideline OECD 471 and EU Method B.13/14.


The test item L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537).


The test was performed in three 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.


 


Experiment 1:


In experiment 1, the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.


The test item showed no precipitates on the plates at any of the concentrations.


The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the presence and the absence of metabolic activation.


The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.


 


Due to an experimental error, no colonies of TA1537 could be observed in any of the tested test item concentrations. Therefore, experiment 1 is repeated as experiment 1b for the bacteria strain TA1537.


 


Experiment 1b:


In experiment 1b, the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 mix in the strain TA1537 using the plate incorporation method.


The test item showed no precipitates on the plates at any of the concentrations.


The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in TA1537. The test item showed no signs of toxicity towards the bacteria strain in both the presence and the absence of metabolic activation.


The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in the strain TA1537, in the presence and the absence of metabolic activation.


 


Experiment 2:


Based on the results of experiment 1 and 1b, the test item was tested up to concentrations of 5000 µg/plate in the presence and absence of S9 mix in all bacteria strains using the pre-incubation method.


The test item showed no precipitates on the plates at any of the test item concentrations.


The bacterial background lawn was not reduced and no decrease in the number of revertants was observed.


The results of this experiment showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the presence and absence of metabolic activation. The test item did not induce a dose-related increase in the number of revertant colonies in all strains, in the presence and absence of metabolic activation.


 


The test item data of experiment 1 for TA1537 are not reported in this report, but the raw data are kept in the test facility in the GLP- archive.


 


Conclusion:


Based on the results of this study it is concluded that L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester 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.