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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:
3.5.2010 - 13.7.2010
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
Report Date:
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 Note Deviation 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): Reaction product of Distillates (petroleum), acid-treated heavy naphthenic and calcium oxide
- 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: solid
- Lot/batch No.: 26.2.2010
- Expiration date of the lot/batch: 26.2.2012
- Stability under test conditions: stable
- Storage condition of test material: The substance was stored in PE bottle at room temperature

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:
50, 150, 500, 1500 and 5000 ug per plate

Vehicle / solvent:
- Vehicle: ethanol p.a.
- Justification for choice of vehicle: the test substance was good suspended in ethanol

Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine hydrochloride monohydrate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
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. Each experiment was repeated. As there was no cytotoxicity, no precipitation or dose-responsiveness, doses in the second experiments remained the same as in the first experiment. In case of toxicity or precipitation, toxic (precipitating) doses would be omitted. In case of mutagenicity, such doses would be chosen which allow construction of a dose-response curve.

Selection of doses/toxicity:
The test substance was suspended in ethanol in the maximum concentration given in guidelines (5000 µg per plate). To obtain homogenous suspension, the mixture was treated by supersonic bath for 30 s.
Homogenous suspension was then obtained which was further diluted by ethanol. The concentration series arisen (10 - 5000 µg per plate) was tested for toxicity in strain TA 100 without metabolic activation. Precipitation and signs of toxicity were not observed in any dose. Particles of the suspension were observable from 1500 µg per plate and the test substance also stained top agar brown. Presence of the particles did not interfere with evaluation.
Therefore, the first mutagenicity experiments were done with the same highest dose as toxicity test. The doses used were 50, 150, 500, 1500 and 5000 µg per plate. As no problems occurred at evaluation with precipitation and toxicity, the same doses were used in the second mutagenicity experiments.

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 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
cytotoxicity was tested with TA98
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
Additional information on results:
Note:
2 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/ In one case (TA 100 without metabolic activation), more than 2 planned experiments were performed, due to more than one formally toxic dose in experiment I. This repetition had no impact on the outcome of the study.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

From the results follows:
TA 100: both experiments without increased values. Negative as well as positive controls are in expected ranges. Conclusion: negative.
TA 1535: both experiments without increased values. Negative as well as positive controls are in expected ranges. Conclusion: negative.
TA 98: both experiments without increased values. Negative as well as positive controls are in expected ranges. Conclusion: negative.
TA 1537: both experiments without increased values. Negative as well as positive controls are in expected ranges. Conclusion: negative.
E. coli WP2 uvrA: both experiments without increased values. Negative as well as positive controls are in expected ranges. Conclusion: negative.

Executive summary:

Test substance was assayed for the mutagenicity by the Bacterial Reverse Mutation Test.

The test was performed according to B.13/14 Mutagenicity – Reverse Mutation Test Using Bacteria, Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008. The method 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 suspended in ethanol and assayed in doses of 50 - 5000 µg which were applied to plates in volumes of 0.1mL.

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

In the arrangement given above, the test substance was nonmutagenic for all the used bacterial strains with as well as without metabolic activation.