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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:
18th February - 26th April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
68308-34-9
Cas Number:
68308-34-9
IUPAC Name:
68308-34-9
Constituent 2
Reference substance name:
Shale oils
EC Number:
269-646-0
EC Name:
Shale oils
IUPAC Name:
269-646-0
Constituent 3
Reference substance name:
Shale oil
IUPAC Name:
Shale oil

Method

Target gene:
Histidine is the target gene for S. typhimurium strains.
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 100, TA 98, TA 97, TA 1535, TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from the livers of adult Sprague-Dawley male rats
Test concentrations with justification for top dose:
0.0001, 0.001, 0.01, 0.05 0.1, 0.25, 0.5, 1 and 5 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (for test substance, vehicle control, 2-nitrofluorene, 9-aminoacridine, 2-acetamidofluorene and water (for sodium azide and mitomycin C)
- Justification for choice of solvent/vehicle: NDA
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535 and TA 100 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 97 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
TA 102 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-acetamidofluorene
Remarks:
TA 98 and TA 100 with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes - 1 hour
- Exposure duration: 48 - 72 hours

NUMBER OF REPLICATIONS: 3 plates

NUMBER OF CELLS EVALUATED: 10^9 cells per ml

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertants

RANGE-FINDING/SCREENING STUDIES:

A preliminary range-linding assay was performed using live tested strains (TA 102, TA 100. TA 98. TA 97 and TA 1535) to determine the optimal non toxic test doses of Shale oils, heavy fraction.

Shale oils, heavy was freshly prepared in DMSO and seven concentrations from range 0.001 - 5.0 mg/plate were tested without and with metabolic activation. An aliquot of the culture was added to 2 ml of molten top agar, along with 0.1 ml ml of the test substance. For the assay with metabolic activation, 0.5 ml of metabolic activation mixture containing 10 % of postmitochondrial traction (S9) together with the bacteria and the test substance were used. The contents were then mixed and poured onto the surface of minimum agar plate. The plates were incubated at 37 ºC for 48-72 hours. Alter the incubation period, the number of revertant colonies per plate was counted.

Cultures were set up in triplicate: negative control and positive control were also included.
Evaluation criteria:
Positive results: concentration-related increase oxer the tested range and reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation. Mf>2. Student's t-test was used for evaluation of statistical significance of mutation frequency increasing against solvent control value.

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:
at concentrations of 0.5 mg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 0.5 mg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 0.5 mg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 0.1 mg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 1 mg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:

Shale oils, heavy in range-finding experiments performed using strains Salmonella typhimurium TA 97, TA 98, TA 1535, TA 102 and TA 100 was tested up to a maximum dose 5.0 mg/plate with and without metabolic activation. This dose was selected according to OECD 471 guideline as the highest tested dose. The concentrations, 5, 1 and 0.5 mg/plate caused reduction of spontaneous revertant counts of all strains of Salmonella typhimurium.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Raw data from the bacterial mutagenicity tests (Tables 1 to 15) are presented in the attached documents.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In the Ames test, with use of live strains of Salmonella Typhimurium (TA 97, TA 98, TA 100, TA 102 and TA 1535) Shale oils, heavy fraction was tested with and without the addition of activation system (S9) for mutagenicity in concentration range from 5 to 0.0001 mg/plate.

The Shale oils heavy fraction produced neither a statistically significant dose-related increase in the number of revertants nor a statistically significant and reproducible positive response at any one of the test points and according these results is considered non mutagenic in this system.
Executive summary:

In the Ames test, with use of live strains of Salmonella Typhimurium (TA 97, TA 98, TA 100, TA 102 and TA 1535) Shale oils, heavy fraction was tested with and without the addition of activation system (S9) for mutagenicity in concentration range from 5 to 0.0001 mg/plate. The experiment was conducted according to OECD guideline 471 and to GLP standard.

The Shale oils heavy fraction produced neither a statistically significant dose-related increase in the number of revertants nor a statistically significant and reproducible positive response at any one of the test points and according these results is considered non mutagenic in this system.