Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30.8.2012 - 9.11.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
3012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Solvent Red 19E
- Physical state: dark viscous liquid/borderline waxy solid
- Analytical purity: 90% (w/w)
- Impurities (identity and concentrations): Solvent Red24 (CAS 85-83-6) 2% (w/w)
- Lot/batch No.: S2409
- Expiration date of the lot/batch: unlisted
- Storage condition of test material: in a glass vial at room temperature

Method

Target gene:
Gene for synthesis of histidine or tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).
Additional strain / cell type characteristics:
other: histidine dependent strain
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).
Additional strain / cell type characteristics:
other: tryptophan dependent strain
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat liver homogenate and mixture of cofactors
Test concentrations with justification for top dose:
I. experiment - 15, 50, 150, 500 and 1500 µg per plate (with and without metabolic activation)
II. experiment - 15, 50, 150, 500 and 1500 µg per plate (without metabolic activation), 50, 100, 250, 500, 1000 µg per plate (with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine, 2-aminofluorene, 2-aminoanthracene, N-methyl-N´-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate incorporation

DURATION
- Exposure duration: 48 – 72 h

SELECTION AGENT (mutation assays): Tryptophan and histidine

NUMBER OF REPLICATIONS: Each test concentration was assessed in triplicate, except for the toxicity test where duplicates were performed.

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth

- OTHER:
Test Procedure
100 µL of test substance of required concentration, 100 µL of 16 – 18 h culture of tester strain, 0.5 mL relevant buffer and 30 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 and kept in a test tube at 45 ± 3 °C. After shaking the mixture was poured into minimal glucose agar plate. After incubation for 48 – 72 hours at 37 ± 1 °C the number of revertant colonies on the plate were counted manually with the exception of positive controls which were counted by an AccuCount 1000. Each experiment was repeated.

Selection of Doses / Toxicity
The test substance was dissolved in the maximum recommended dose (5000 µg/0.1 mL) in DMSO. It did not succeed. The maximum of the test substance dissolved in DMSO was less than 1000 µg/0.1 mL. Toxicity was tested in concentration series 10 – 1000 µg/plate, completed by the dose of 5000 µg per plate (nominal values, undissolved). This concentration series was then tested for toxicity in strain TA 100 without metabolic activation.
Turbidity appeared from the dose 200 µg per plate and the test substance made a homogenous film on the surface of the top agar. The test substance was not toxic at any dose and evaluation was possible in all doses.
The test substance is soluble in acetone and in small amounts is comparable with the test so the toxicity test was repeated with the test substance dissolved in this solvent. The test substance was dissolved in acetone at the concentration 100 mg/mL (10 mg/0.1 mL) and a concentration series (20 – 10 000 µg/0.1 mL) were made by dilution. From these applications forms, 50 µL was dosed per plate (doses 10 – 5000 µg/plate). After adding to top agar, the test substance cleaved at tube walls starting with concentration of 1000 µg/plate. On plates, the test substance was observable as a black non-homogeously spread bubbles and mist. In addition, acetone itself decreased the number of revertants with regard to spontaneous reversion. Even if the test substance was not toxic at any dose, testing in acetone was disclaimed and in the end the test substance was diluted in DMSO.
Tee dose of 1500 µg/plate was therefore used as maximum for the first mutagenicity experiments. The maximum dose was diluted accordingly to five different analysable concentrations with approximate half-log intervals between test points. There were no problems with toxicity or with evaluation. In some experiments mutagenicity was decreased or stagnated at the highest dose. The second experiments with metabolic activation were modified to obtaining better dose-response dependence from 50 to 1000 µg/plate.
Fresh solutions of test substance were prepared before each experiment. All concentrations of the test substance solution were dosed in the volume of 0.1 mL/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. 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 “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 not a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
According to OECD Test Guideline 471, the biological relevant of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.


References:
1. Maron D. M., Ames B. N. (1983): Revised methods for the Salmonella mutagenicity test, Mutat. Res. 113, 173 - 215
2. 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 - 240

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
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 98
Metabolic activation:
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
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 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
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 1535
Metabolic activation:
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 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:
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:
The results obtained in most experiments without metabolic activation did not show substantial (biologically significant) increases in the number of revertants versus solvent controls (Rt / RC < 2) and no experiment gave evidence of rising trend in the number of revertants with increasing dose.
Neither increasing of number of revertants nor dose-dependence was observed in experiments with S. typhimurium TA 1537 and E. coli WP2 uvrA with metabolic activation.
Increased values of numbers of revertants as well as dose-dependence were observed in three Salmonella strains with metabolic activation. These results were confirmed with repeated experiments.

COMPARISON WITH HISTORICAL CONTROL DATA:
All the control numbers were compared with historical ranges of mutant frequencies obtained in VUOS laboratory. The actual numbers were in ranges of the historical numbers.

Applicant's summary and conclusion

Conclusions:
The test material was mutagenic for tester strains S. typhimurium TA 100, TA 98 and TA 1535 with metabolic activation.

The test substance was non-mutagenic for all the S. typhimurium as well as the E. coli strain without metabolic activation, and for S. typhimurium TA 1537 and E. coli WP2 uvrA with metabolic activation.
Executive summary:

Test material was assessed for mutagenicity according to 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 material was dissolved in DMSO and assayed in doses of 15-1500 µg, 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 test substance was mutagenic for tester strains S. typhimurium TA 100, TA 98 and TA 1535 with metabolic activation.

The test substance was non-mutagenic for all the used tester strains without metabolic activation as well as for S. typhimurium TA 1537 and E. coli WP2 uvrA with metabolic activation.