Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 06, 2007 - December 18, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Official Journal of the European Communities L136, 8. June 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
HIS operon (S. thyphimurium)
TRP operon (E. coli)
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 98
Details on mammalian cell lines (if applicable):
his D 3052, uvrB, rfa + R-factor
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
S. typhimurium TA 100
Details on mammalian cell lines (if applicable):
his G 46, uvrB, rfa + R-factor
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
S. typhimurium TA 102
Details on mammalian cell lines (if applicable):
his G 428, rfa + R-factor
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
S. typhimurium TA 1535
Details on mammalian cell lines (if applicable):
his G 46, uvrB, rfa
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
S. typhimurium TA 1537
Details on mammalian cell lines (if applicable):
his C 3076, uvrB, rfa
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
E. coli WP2 uvr A pKM 101
Details on mammalian cell lines (if applicable):
uvrA pkM101
Additional strain characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Test concentrations with justification for top dose:
The test material concentrations used were selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1st series: 0.5, 1.58, 5, 15.8, 50, 158 and 500 μg per plate
2nd series: 15.8, 28.1, 50, 88.9 and 158 μg per plate
In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively.
Vehicle:
Acetone
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
cumene hydroperoxide
other: daunomycin
Remarks:
without S9 mix
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
with S9 mix
Details on test system and conditions:
The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

Mean Number of Colonies Maximal Mean Number of Colonies over
(Solvent Control) the Actual Solvent Control (Test Material)
----------------------------------------------------------------------------------------------
<=10 <=9 >=30
<=30 <=19 >=40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200
Assessment: No increase Clear increase
----------------------------------------------------------------------------------------------

All further results, ranging between "no" and "clear", are assessed as "weak in-creases".

Interpretations:

A test material is defined as non-mutagenic in this assay if:
- "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if:
- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.
Evaluation criteria:
For details see results.
Statistics:
n.a.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes

Applicant's summary and conclusion

Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.