Registration Dossier

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:
Feb 06 - Mar 08, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD TG 471.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Method

Target gene:
HIS Operon (Salmonella typhimurium), TRP operon (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Standard I Nutrient Broth (Merck KGaA, Darmstadt, Germany)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: Standard I Nutrient Broth (Merck KGaA, Darmstadt, Germany)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
by liver S9 mix obtained from rats pre-treated with Aroclor 1254
Test concentrations with justification for top dose:
1st series: 5.00, 15.8, 50.0, 158, 500, 1580, and 5000 µg/plate
2nd series: 15.8, 50.0, 158, 500, 1580, and 2810 µg/plate
Vehicle / solvent:
DMSO
Controls
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: Daunomycin, 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
The incubation of plates was performed at (37 +/- 1) °C for 2 to 3 days.

NUMBER OF REPLICATIONS: 3

Evaluation criteria:
Definitions:
The assessment of test material-induced effects is dependent on the number of spontaneous re-vertants 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 crite-ria, based upon the historical controls of the laboratory and statistical considerations, are estab-lished:

Mean Number of Colonies
(Solvent Control) Maximal Mean Number of Colonies over the Actual Solvent Control (Test Material)
≤ 10 ≤ 9 ≥ 30
≤ 30 ≤ 19 ≥ 40
≤ 80 ≤ 29 ≥ 80
≤ 200 ≤ 49 ≥ 120
≤ 500 ≤ 99 ≥ 200
Assessment: "No Increase" "Clear Increase"
All further results, ranging between "no" and "clear", are assessed as "weak increases".

A test material is defined as negative or non-mutagenic in this assay if
• the assay is considered valid, and
• "no" or "weak increases" occur in the test series performed. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as positive or mutagenic in this assay if
• the assay is considered valid and
• a dose dependent (over at least two test material concentrations) increase in the number of re-vertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system or
• "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.
There is no requirement for verification of a clear positive response.
In all further cases, a third test series with the bacterial strain in question should be performed and the results of each series should be discussed on a case by case basis.

Statistics:
not performed

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentration larger or equal to 2810 µg/plate
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 and without metabolic activation

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

Study Design

This GLP study was performed according to OECD TG 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254 -pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the 1st and 30 % in the 2nd series, respectively.

Results

The test material was dissolved in DMSO and tested at concentrations ranging from 5.00 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations ≥ 2810 µg/plate. Toxicity to the bacteria was not observed.
Daunomycin, sodium azide, 9 -aminoacridine, 4 -nitroquinolin-N-oxide served as strain specific positive control test materials in the absence of S9 mix. 2 -Aminoanthracene was used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no clear increases in the number of revertants of any bacterial strain.

Therefore the test material was not mutagenic in this assay.

Conclusion

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