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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19.7.2010 - 21.9.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
see Deviations in the Section: Additional information on results
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tar, brown-coal, low-temp.
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Smiles notation: not known - UVCB substance
- InChl: not known - UVCB substance
- Substance type: technical product
- Physical state: viscous liquid
- Lot/batch No.: T201E
- Expiration date of the lot/batch: 31.3.2011
- Stability under test conditions: stable
- Storage condition of test material: The substance was stored in closed vessel in dry room at the temperature bellow 25ºC in dark

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine dependent strain
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: tryptophane dependent strain
Metabolic activation:
with and without
Metabolic activation system:
a supernatant of rat liver and a mixture of cofactors
Test concentrations with justification for top dose:
5, 15, 50, 150, 500 µg per plate
(in select strains also 1.5, 300 µg per plate)
Selection of doses:
The test substance was dissolved in DMSO in concentration 50 mg/mL from what 0.1 mL was applied onto plates in maximum dose (5 mg per plate) given in guidelines. The highest concentration was further diluted to origination of a concentration series (10-5000 µg per plate), which was tested for toxicity in strain TA 100 without metabolic activation.
Cytotoxicity was observed from the dose of 500 µg per plate (thinning of bacterial background). As number of revertants in solvent control decreased considerably in comparison with spontaneous reversion, toxicity experiment was repeated in TA 98 with lower doses from 10 to 1000 µg per plate with the same result in fact.
Dose of 500 µg per plate was then used as maximum in the first mutagenicity experiments. The starting concentration was diluted according to guidelines (five different analysable concentrations with approximately half log (i.e. √10) intervals between test points). The maximum dose caused clouding of top agar and it was partially toxic (thinning or absence of background) in some experiments. In the second experiments was therefore used additional dose of 300 µg per plate in some experiments.




Vehicle / solvent:
- Vehicle: dimethylsulfoxide
- Justification for choice of vehicle: optimal solvent for test substance

Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethylsulfoxide
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethylsulfoxide
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethylsulfoxide
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethylsulfoxide
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethylsulfoxide
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine hydrochloride monohydrate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethylsulfoxide
Positive controls:
yes
Positive control substance:
other: N-methyl-N´-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
THE BACTERIAL TESTER STRAINS:
- histidine dependent Salmonella typhimurium TA 98 (CCM 3811), TA 1535 (CCM 3814), and tryptophan dependent strain Escherichia coli WP2 uvrA (CCM 4751) were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno,
- TA 100 (CIP 103796, lot. No.1008) and TA 1537 (CIP 103799, lot No. 34508) were from Biological Resource Center of Institut Pasteur (CRBIP), Paris.
Strains TA 1537 and TA 98 detect frame shift mutations,
strains TA 100 and TA 1535 serve to detect base-pair substitution mutations,
strain E.coli WP2 uvrA detects cross-linking mutagens.

METHOD OF APPLICATION: in agar (plate incorporation)

Plate incorporation test
Test procedure:
100 µL of the test substance in required concentration, 0.1 mL 16-18 h culture of tester strain, 0.5 mL relevant buffer and 30 or 100 µL of S9 postmitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL top agar (with trace of histidine or tryptophan) kept in a test tube at 45±3ºC. After shaking the mixture was poured into a minimal glucose agar plate. After incubation of 48 - 72 h at 37±1ºC, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted automatically.
For an adequate estimate of variation, triplicate plating was used at each dose level.

Selection of doses/toxicity:
The test substance was dissolved in DMSO in concentration 50 mg/mL from what 0.1 mL was applied onto plates in maximum dose (5 mg per plate) given in guidelines. The highest concentration was further diluted to origination of a concentration series (10-5000 µg per plate), which was tested for toxicity in strain TA 100 without metabolic activation. Application forms were clear to 500 µg per plate; starting with the dose of 500 µg per plate application forms were clouded (emulsion).
Toxicity was observed from the dose of 500 µg per plate ( thinning of bacterial background). As number of revertants in solvent control decreased considerably in comparison with spontaneous reversion, toxicity experiment was repeated in TA 98 with lower doses from 10 to 1000 µg per plate with the same result in fact.
Dose of 500 µg per plate was then used as maximum in the first mutagenicity experiments. The starting concentration was diluted according to guidelines (five different analysable concentrations with approximately half log (i.e. √10) intervals between test points). The maximum dose caused clouding of top agar and it was partially toxic (thinning or absence of background) in some experiments. In the second experiments was therefore used additional dose of 300 µg per plate in some experiments. Third experiment in TA 1537 with metabolic activation was performed due to of prospecting of dose effect gain with dose range of 1.5-150 µg per plate.


Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule that is compatible with the application of statistical methods (see Statistics - litarature). 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.

Statistics:
Literature:
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
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
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
tested only without metabolic activation: toxicity > 500 µg/plate without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
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
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxicity > 500 µg/plate without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
other: weak mutagen
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
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
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
other: weak mutagen
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Deviations:
1/ In case of the strain TA 98, frozen stock culture after expiration of minimum shelf life (two years) was used for experiments - a deviation from SOP. (Standard operation procedure). The bacteria were tested for their properties (phenotype confirmation, response to positive controls). All the tested properties were in expected ranges therefore the use of this culture had no impact on the outcome of the study.
2/ More than two planned experiments were performed in TA 1537 due to slight growth of bacteria. Third experiment was done due to clarification of prospective dose-effect dependence. More than two planned experiments were performed also in TA 98 with metabolic activation, due to solvent control out of historical controls in the second experiment. Results of this experiment are not taken into account. Repetition of experiments had no impact on the outcome of the study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation in all used strains
negative with metabolic activation in TA 100, TA 1535 and WP2 uvrA strains
other: weakly mutagenic with metabolic activation in TA 1537 strain
other: weakly mutagenic with metabolic activation in TA 98 strain
Executive summary:

Test substance - Tar, brown-coal, low-temp. - was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed 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 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used.

Selection of doses: 
The test substance was dissolved in DMSO in concentration 50 mg/mL from what 0.1 mL was applied onto plates in maximum dose (5 mg per plate) given in guidelines. The highest concentration was further diluted to origination of a concentration series (10-5000 µg per plate), which was tested for toxicity in strain TA 100 without metabolic activation.

Cytotoxicity was observed from the dose of 500 µg per plate (thinning of bacterial background). As number of revertants in solvent control decreased considerably in comparison with spontaneous reversion, toxicity experiment was repeated in TA 98 with lower doses from 10 to 1000 µg per plate with the same result in fact. 
Dose of 500 µg per plate was then used as maximum in the first mutagenicity experiments. The starting concentration was diluted according to guidelines (five different analysable concentrations with approximately half log (i.e. √10) intervals between test points). The maximum dose caused clouding of top agar and it was partially toxic (thinning or absence of background) in some experiments. In the second experiments was therefore used additional dose of 300 µg per plate in some experiments.

The test substance was dissolved in dimethylsulfoxide and assayed in doses of 1.5 - 500 µg which were applied to plates in volume of 0.1 mL.

Two series of experiments were performed with each strain, without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

Results:

In the arrangement given above, the test substance was nonmutagenic for all the used tested strains without metabolic activation and for strains Salmonella typhimurium TA 100, TA 1535 and Escherichia coli WP2 uvrA with metabolic activation as well. The test substance was weakly mutagenic for strain TA 1537 with metabolic activation and for strain TA 98 with metabolic activation.