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Toxicological information

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:
Nov 19 - Dec 05, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

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:
solid: bulk

Method

Target gene:
HIS Operon (Salmonella typhimurium), TRP operon (E. coli)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : obtained liver S9 mix obtained from male Wistar rats pre-treated with Aroclor 1254
- method of preparation of S9 mix: intraperitoneal injection of Aroclor 1254; on day 5 to 7 animals were sacrificed, livers were removed and homogenized; homogenate was spun at 9000 x g for 10 minutes; supernatant fluid was decanted and stored at - 196°C
- concentration or volume of S9 mix and S9 in the final culture medium: 10% and 30% in the first and second series, respectively
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): metabolic activity
Test concentrations with justification for top dose:
1st series: 0.5, 1.58, 5.00, 15.8, 50.0, 158, and 500 µg/plate
2nd series: 15.8, 28.1, 50.0, 88.9, and 158 µg/plate
Vehicle / solvent:
Acetone- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: standard vehicle with no influence on the number of spontaneous revertants of any strain

Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
benzo(a)pyrene
cumene hydroperoxide
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

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; reduction in the number of spontaneous revertants


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 criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

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".
Interpretations:
A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the test series performed. ("Weak increases" randomly oc-cur 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 rever-tants 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 and the results of each series be discussed case by case.

Statistics:
not performed

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: at concentration larger or equal to 50 µg/plate

RANGE-FINDING/SCREENING STUDIES: In the first experiment seven concentrations ranging from 0.5 to 500 µg/plate were tested. Due to precipitation and the absence of cytotoxicity the following test material concentrations were used:
0.5, 1.58, 5.0, 15.8, 50.0, 158.0 and 500.0 µg/plate (first experiment)
15.8, 28.1, 50.0, 88.9 and 158 µg/plate (second experiment)

Ames test:
- Signs of toxicity : none
- Mean number of revertant colonies per plate and standard deviation: see attached document 1

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: The positive control should induce a "clear increase" in the number of revertants (see attached document 2)

- Negative (solvent/vehicle) historical control data:
TA 98: 15 - 60
TA 100: 75 - 200
TA 102: 200 - 450
TA 1535: 3 - 37
TA 1537: 4 - 31
WP2 uvrA: 10 -70

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.
Executive summary:

This GLP study was performed according to OECD TG 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA102, 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.

The test material was dissolved in acetone and tested at concentrations ranging from 0.50 to 500 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations ≥ 158 µg/plate. Toxicity to the bacteria was not observed.
Daunomycin, sodium azide, 9 -aminoacridine, 4 -nitroquinolin-N-oxide and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2 -Aminoanthracene and benzo[a]pyrene were 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.
With and without addition of S9 mix as the external metabolizing system, the test material did not induce any clear or dose dependent increase in the mutation rates under the experimental conditions described.

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