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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:
03 Aug. - 06 Sep. 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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): anthracene oil (> 50 ppm BaP)
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Substance type: organic
- Physical state: liquid
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- Storage condition of test material: room temperature, exclusion of light

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
Microsomal fraction prepared from induced livers of male Wistar rats, induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) orally (3x)
Test concentrations with justification for top dose:
1st experiment: 3.16, 10, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (TA 98, TA 100, +/-S9)
1.0, 3.16, 10, 31.6, 100, 316, and 1000 µg/plate (TA 1535, TA 1537, - S9)
10, 31.6, 100, 316, 1000 and 2500 µg/plate (TA 1535, TA 1537, + S9)
3.16, 10, 31.6, 100, 316, and 1000 µg/plate (TA 102, +/-S9)
2nd experiment: with variations based on results of 1st experiment (Report, p. 11)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compatible with survival of bacteria and S9 activity
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: see Report p. 15
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth/colony formation

Evaluation criteria:
Considered as mutagenic
- if a clear and dose-related increase in the number of revertants occurs in at least one tester with or without metabolic activation
and/or
- if a biologically relevant positive response for at least one of the dose groups occurs in at least one tester with or without metabolic activation.

An increase is considered relevant
- if in TA 100 and TA 102 mutation rate is at least twice as high as the rate of the solvent control;
- if in TA 98, TA 1535, and TA 1537 the mutation rate is at least 3x higher than that of the solvent control.


Statistics:
According to the OECD guidelines, the biological relevance is the criterion for the interpretation of the results: a statistical evaluation was not considered necessary under this premise (report p. 21).

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at >= 31.6 µg/pl. (-S9); at >= 316 µg/pl. (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
not or weakly cytotoxic in the mutagenic concentration range
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 102
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
weak positive effect, not reproducible
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
not in the relevant mutagenic concentration range
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 98
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
dose response positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
not cytotoxic in the relevant concentration range
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Summary:

The positive effects noted without S9 were weak and not reproducible in either experiment (see below).

In the presence of S9 clear dose-related increases in the mutation rates were found in TA 98 and 1537 and a weak positive correlation in TA 100 and TA 102, reproducible at non-cytotoxic concentrations in both experiments. The most responsive tester strain was TA 98.

[Mutation factors (+S9) = 3 - 14x above background with maximum in TA 98]

In either experiment, no biological relevant increases in the mutation rate were seen in TA 1535.

Experiment I:

No relevant increases in TA 98, TA 100, TA 1537, and TA 102 (-S9);

relevant increases in TA 98, TA 100, and TA 1537 (>= 10 µg/plate, +S9)

Experiment II:

Relevant increases in TA 98 (>= 15 µg/plate, -S9);

relevant increases in TA 98, TA 100, TA 1537, and TA 102 (>= 5 - 500 µg/plate, +S9)

Dose-response relationship of the reversion rate was observed with each tester strain.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive