Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From September 20 to November 07, 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Reliability of the source study is 1

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Acid Brown 161:1 - Similar Substance 02
IUPAC Name:
Acid Brown 161:1 - Similar Substance 02
Test material form:
solid: particulate/powder

Method

Target gene:
histidine or tryptophan locus
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 fraction of rat liver
Test concentrations with justification for top dose:
100, 230, 500, 1000, 2300 µg (applied to plates in volume of 0.1 mL)
Vehicle / solvent:
- Vehicle/solvent used: water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
other: 4-nitro-o-phenylenediamine, 2-aminofluorene, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation) in the first experiment and pre-incubation in the second experiment.

DURATION
- Preincubation period: 30 min in the second experiment
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: triplicate plating was used at each dose level except in the toxicity test with strain TA 98, where test substance was tested in duplo

FIRST EXPERIMENT
100 µL of the test substance of required concentration, 100 µL of 16-18 h culture of tester strain of density 10ʌ8-10ʌ9 CFU/mL, 0.5 mL relevant buffer and 30 (100) µL of S9 post mitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45 ± 3 °C. After shaking the mixture was poured into a minimal glucose agar plate.

SECOND EXPERIMENT
0.5 mL of relevant buffer, 100 µL of the test substance of required concentration, 100 µL of 16-18 h culture of tester strain of density 10ʌ8-10ʌ9 CFU/mL and 30 (100) µL of S9 post mitochondrial fraction in experiment with metabolic activation were mixed and shaken at 37 ± 1 °C for 30 minutes. Then, 2 mL of molten top agar (with trace of histidine or tryptophan) was added and the mixture was poured into a minimal glucose agar plate.Petri dishes prepared one or the other way were incubated of 48 - 72 h at 37 ± 1 °C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000.

SELECTION OF DOSES/TOXICITY
2.0 mL of water for injection were added to 100 mg of the test substance in a test tube to reach the maximum dose recommended in guidelines - 5000 µg per plate (per 0.1 mL). The test substance was not dissolved at the highest concentration of application form; particles of the test substance were observed on Petri dishes in concentrations 2500 and 5000 µg per plate. Some particles were observed also in the concentration of 1000 µg per plate where besides particles on the agar surface other particles were contained in the agar. For toxicity experiment, the starting suspension (5000 µg/0.1 mL) was diluted to concentration series 10 - 5000 µg per plate. The concentration row was tested for toxicity in strain TA 98 without metabolic activation. Petri dishes were coloured red. In the highest concentration (5000 µg/0.1 mL) the test substance in the top agar disabled evaluation, so some colonies could be omitted. No toxicity was observed in any dose. The concentration of 2300 µg/0.1 mL, which sponsor declared as maximum solubility in water, was then used as maximum in the first mutagenicity experiments. No mutagenicity either toxicity was observed in the first experiments, precipitation in the top agar was observed in the maximum dose, so the same concentrations were used for the second mutagenicity experiments. The second mutagenicity experiments were performed with pre-incubation.Fresh solutions of the test substance were prepared before each experiment. Concentrations of the test substance solution were dosed in the volume of 0.1 mL per plate.
Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods (2, 3). After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached. An increase is considered as „biologically relevant“:
-if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
-if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;

A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Statistics:
According to OECD TG 471, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA strain.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Non mutagenic
Executive summary:

Method

The test substance was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to the EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria,which is analogous to the OECD Guideline 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was diluted in water for injection and assayed in doses of 100 - 2300 mg per plate, which were applied to plates in volume of 0.1 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors. The first experiments were performed without and the second experiments with 30 minutes of pre-incubation at 37±1 °C and shaking.

 

Results

In the arrangement given above, the test substance, was non-mutagenic for all the used tester strains without as well as with metabolic activation. Pre-incubation had no influence on study results.