Registration Dossier

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
Remarks:
OECD 471

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted 21. Jul. 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No. 440/2008.
Adopted 30. May 2008
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Details on test material:
The test material is a UVCB substance.
Specific details on test material used for the study:
TEST ITEM
Name ACID BROWN 396:1
Batch no. 77171
Appearance Brown powder
Purity 100% (UVCB)
Homogeneity homogeneous
Expiry date 18 Sep. 2020
Storage Room Temperature (20 ± 5 °C)
EINECS-No. 947-779-0


STORAGE
The test item was stored in the test facility in a closed vessel at room temperature (20±5°C).

Method

Species / strainopen allclose all
Species / strain / cell type:
other: S. typhimurium TA97a
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Vehicle / solvent:
-DMSO
-Deminaralised water
Controls
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO), batch: 187256959, for the positive controls nitro-phenylendiamine, benzo-a-pyrene and 2-amino-anthracene and batch: 218270919 for the test item. Demineralised water, batch: 20180614 for the positive control sodium azide
Positive controls:
yes
Remarks:
All mutagenic substances were stored as ready to use solution in the test facility in the deep freezer. The respective positive control solution was thawed on the day of each experiment.
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene Diamine 2-Amino-Anthracene
Details on test system and experimental conditions:
CULTURE OF BACTERIA
Species Salmonella typhimurium LT2
Strains TA97a, TA98, TA100, TA102 and TA1535
All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH and were stored as lyophilizates in the refrigerator at 2-8 °C. The lyophilizates were used to prepare permanent cultures which were filled into vials and stored at < - 75 °C.
Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutrient broth. After incubation for eight hours at 37 ±1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

TEST VESSELS
All vessels used are made of glass or sterilizable plastic. They were sterilized before use by autoclaving.
The following vessels were used:
- Schott-bottles, glass vials, and culture flasks for solutions and media;
- Plastic petri plates;
- Test tubes for top-agar-bacteria-substance mix.

PREPARATIONS
Different media and solutions were prepared preliminary. On the day of the test, the bacteria cultures were checked for growth visually. The incubation 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.

EXPERIMENTAL PARAMETERS
First Experiment:
Date of treatment 09. Oct. 2018
Concentrations tested 5000 / 1500 / 500 / 150 / 50 µg/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tested strains TA97a, TA98, TA100, TA102, TA1535
Method plate incorporation method

Second Experiment:
Date of treatment 16. Oct. 2018
Concentrations tested 5000 / 2500 / 1250 / 625 / 313 / 156 µg/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tested strains TA97a, TA98, TA100, TA102, TA1535
Method pre-incubation method

DESCRIPTION OF THE METHOD
Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used.
For the top agar, agar basis was melted in a microwave oven, then the histi-dine-biotin-solution was added and 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:
- test solution at each dose level, solvent (negative control) or reference mu-tagen solution (positive control);
- S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation);
- bacteria suspension;
- 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.
This method was used to prepare samples of experiment I.

PRE-INCUBATION METHOD
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
- test solution at each dose level, solvent (negative control) or reference mu-tagen solution (positive control);
- S9 mix (for test with metaboli activation) or phosphate buffer (for test without metabolic activation);
- bacteria suspension.
After the pre-incubation for 20 minutes, top agar was added and the tube was gently vortexed. 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.
This method was used to prepare samples of experiment II.
Evaluation criteria:
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 deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants less mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: S. Typhimurium TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Additional information on results:
FIRST EXPERIMENT
All strains met the criterion of at least 10^9 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
In the first experiment, the test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the conditions of this experiment. To verify this result, a further experiment was performed.

SECOND EXPERIMENT
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the nega-tive controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all were within the historical control data ranges. In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial back-ground lawn was visible and not affected. The number of revertant colonies was not re-duced. No increase of the number of revertant colonies in the treatments with and without meta-bolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test item is stated as not mutagenic under the conditions of this experiment.

MUTAGENICITY OF TEST ITEM
The test item ACID BROWN 396:1 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 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 ACID BROWN 396:1 is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

ACCEPTABILITY OF STUDY, DISCUSSION
In all experiments, no precipitation of the test item ACID BROWN 396:1 was observed at any of the tested concentrations up to 5000 µg/plate.
In the first and the second experiment, the test item caused no cytotoxicity towards all bacteria strains.
The confirmation tests of the genotype did not show any irregularities. 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, but all were in-creased 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.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that ACID BROWN 396:1 is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.
Executive summary:

Two valid experiments were performed.

The test item ACID BROWN 396:1 was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535) and the test was performed in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

Experiment 1:

In the first experiment, the test item (dissolved in DMSO) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 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 absence and presence 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.

Experiment 2:

Based on the results of the first experiment, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence 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 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 absence and presence of metabolic activation.

The results of this experiments 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 absence and presence of metabolic activation.

In both the experiments, the test item ACID BROWN 396:1 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 indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Since all criteria for acceptability have been met, the study is considered valid.

Based on the results of this study it is concluded that ACID BROWN 396:1 is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions.