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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:
17 Jul. - 03 Sep. 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline 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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
Plate incorporation test
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Pitch, coal tar, high-temp., < 1% 4- to 5-membered condensed ring aromatic hydrocarbons
EC Number:
701-305-8
Molecular formula:
not applicaple - UVCB
IUPAC Name:
Pitch, coal tar, high-temp., < 1% 4- to 5-membered condensed ring aromatic hydrocarbons
Test material form:
solid: pellets

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other:
Remarks:
see report, p. 15 for details
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction was 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:
Pre-experiment: 3.16, 10, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (TA 98, TA 100: +/-S9)
1st experiment: 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (all tester strains, +/-S9)
2nd experiment: 250, 500, 1000, 2000, 2500, and 5000 µg/plate (all tester strains except TA 1537, +/-S9),
in addition: 125 µg/plate only for TA 98, +S9,
20, 80, 200, 800, 2000, and 5000 µg/plate (TA 1537, +S9),
250, 500, 1000, 2000, 3500, and 5000 µg/plate (TA 1537, -S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Test item was suspended in ethanol and diluted prior to use.
- Justification for choice of solvent/vehicle: compatible with survival of bacteria and S9 activity
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Sodium azide, 4-nitro-o-phenylene-diamine (4-NOPD), methyl methane sulphonate (MMS), 2-aminoanthracene (2-AA); see Report p. 15 for details
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation): Materials were mixed in test tube and poured over the surface of a minimal agar plate
- Test solution (at each dose level, solvent control, negative control, or positive control: 100 µl
- S9 mix (for testing with metabolic activation or S9 mix substitution buffer (for testing without metabolic activation): 500 µl
- Bacteria suspension: 100 µl
- Overlaying agar: 2000 µl

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
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
reproducible, dose-response relationship in both experiments with metabolic activation. Without S9, weakly positive in one test.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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
Remarks:
reproducible
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
reproducible, dose-response relationship
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in all tester strains at test concentrations of >= 1000 µg/plate (1st experiment);
in all tester strains except tester strain TA 1537 at test concentrations of >= 1000 µg/plate (2nd experiment)
in tester strain TA 1537 at a test concentration of >= 1000 µg/plate (2nd experiment, without metabolic activation) and
at a test concentration of >= 800 µg/plate (2nd experiment, with metabolic activation)


Any other information on results incl. tables

Maximum mutation factor: 14.3 (tester strain TA 98 with metabolic activation at a dose of 3500 µg/plate, 1st experiment)

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
positive