Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04.04. - 19.05.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: particulate/powder

Method

Target gene:
gene for histidine or tryptophan synthesis
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
supernatant of rat liver and a mixture of cofactors.
Test concentrations with justification for top dose:
50, 150, 500, 1500, 5000 μgSelection of doses/toxicity: : Sponsor declared solubility in water 50 g per litre (at 25°C) what is the same concentration recommended as maximum concentration in OECD TG 471 for this assay. The test substance was then dissolved in water for injection till the maximum recommended concentration 5000 μg per 0.1 mL.
Vehicle / solvent:
Water for injection, Ardeapharma, Lot. No.: 1508310536- Justification for choice of solvent/vehicle: solubility of the substance
Controls
Untreated negative controls:
yes
Remarks:
Dimethyl sulfoxid
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylenediamine, 2-aminofluorene, 2-aminoanthracene, N-methyl-N´-nitro-N-nitrosoguanidine, 9-aminoacridine hydrochloride monohydrate
Details on test system and experimental conditions:
The bacterial tester strains Salmonella typhimurium TA 1535 (CCM 3814, lot. No. 2101200916917), TA 98 (CCM 3811, lot No. 01022001220053), TA 100 (CCM 3812, lot No. 0102201220054) and TA 1537 (CCM 3815, lot No. 2101200916918) as well as Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732) were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno.Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagensMETHOD OF APPLICATION: in agar (plate incorporation)NUMBER OF REPLICATIONS: two seriesDETERMINATION OF CYTOTOXICITY- Method: relative total growth
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 doseresponse effect occurs and/or a doubling of the ratio Rt/Rc is reached.
Statistics:
For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the application of statistical methods:1. Maron D. M., Ames B. N. (1983): Revised methods for the Salmonella mutagenicity test, Mutat. Res. 113, 173 - 2152. Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 - 4173. Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 – 91

Results and discussion

Test resultsopen allclose all
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 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
positive
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 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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
Additional information on results:
HISTORICAL CONTROL DATAEach experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 mL of DMSO. All the control numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers. ADDITIONAL INFORMATION ON CYTOTOXICITY:No toxicity was observed in any dose. Precipitation in the top agar occurred from 2500 μg.0.1mL-1. The occurrence of precipitation in top agar was then investigated in the test substance concentration of 1500 μg.0.1mL-1. This concentration of the test substance did not cause precipitation in top agar so concentrations for the first mutagenicity experiments were chosen as 5000, 1500, 500, 150 and 50 μg per plate.

Applicant's summary and conclusion

Conclusions:
Under the above-described experimental design, the test substance, Acid Black 227, was mutagenic for the Salmonella typhimurium TA 98, TA 1537 and TA 100 in experiments with as well as without metabolic activation. The test substance was non-mutagenic for Escherichia coli and S. typhimurium TA 1535 with as well as without metabolic activation.
Executive summary:

The test substance Acid Black 227 was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 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 50 - 5000 µg per plate, which were applied to plates in volume of 0.1 mL.

The first mutagenicity experiments were performed without and with metabolic activation using a supernatant of rat liver (30 μL or 100 μL per plate) and a mixture of cofactors by the plate incorporation test with a dose range of 50 – 5000 µg per plate.

To modify experimental conditions and to increase the sensitivity of the assay, the second mutagenicity experiments were performed with pre-incubation.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

In the arrangement given above, the test substance, Acid Black 227, was markedly mutagenic for the Salmonella typhimurium TA 98, TA 1537 in experiments with as well as without metabolic activation and slightly mutagenic for TA 100 rather without metabolic activation/preincubation. The test substance was non-mutagenic for Escherichia coli and S. typhimurium TA 1535 with as well as without metabolic activation.