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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:
Between 18 July 2000 and 08 September 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: pure active substance
- Analytical purity: 98.1%
- isomer composition: ca. 10-12 % cis- and 86-88 % trans isomers
- Lot/batch No.: F02214-01D
- Storage condition of test material: room temperature under nitrogen in the dark

Method

Target gene:
Not Applicable for Ame's test
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 derived from rat Sprague-Dawley
Test concentrations with justification for top dose:
See Table 2 TEST COMPOUND CONCENTRATIONS USED in "Any other information on results incl. tables".
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (dried on molecular sieve)
- Justification for choice of solvent/vehicle: to improve water solubility of the test substance. Well known solvent/vehicle not reacting with the test substance. Volume of vehicle/solvent in the medium: not a requirement of the test guideline.
Vehicle/solvent controls (negative control) were tested in the current study.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
3 µg/plate for TA100 and 5 µg/plate for TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 μg/plate for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.5 μg/plate for TA102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 μg/plate for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 μg/plate for TA98 (with metabolic activation (+S9))
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxyanthraquinone (DANTHRON)
Remarks:
10 μg/plate for TA102 (with metabolic activation (+S9))
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, 10 μg/plate for WP2uvrA (all with metabolic activation (+S9))
Details on test system and experimental conditions:
DURATION
- Preincubation period: Not Applicable (NA)
- Exposure duration: NA
- Expression time (cells in growth medium): ca. 48 hours at 37°C
- Selection time (if incubation with a selection agent): NA
- Fixation time (start of exposure up to fixation or harvest of cells): NA


SELECTION AGENT (mutation assays): NA
SPINDLE INHIBITOR (cytogenetic assays): NA
STAIN (for cytogenetic assays): NA


NUMBER OF REPLICATIONS: NA


NUMBER OF CELLS EVALUATED: NA


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (Preliminary cytotoxicity test (see Table 2a in the field "Any other information on material & methods including tables"))


OTHER EXAMINATIONS:
- Determination of polyploidy: NA
- Determination of endoreplication: NA
- Other: NONE


OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay was considered valid if the following criteria were met:
1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (according to historical control 1998 & 1999).
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the approximate range of 1 to 9.9 billion bacteria per ml.
4. Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
5. There should be a minimum of four non-toxic test material dose levels.
6. There should be no evidence of excessive contamination.
Evaluation criteria:
EVALUATION CRITERIA: The test material may be considered positive in this test system if the following criteria are met: the test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression if necessary

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 ug/plate for TA100
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: NA
- Effects of osmolality: NA
- Evaporation from medium: Test material vapour pressure (0.02 Pa) is too low to expect a significant effect of evaporation on test results. Test material is not classified as volatile according to EPA (EPA's criteria: Henry LC > 0.00001 atm m3/mol & MW< 200 g/mol) as Henry LC = 0.0000005 atm m3/mol (EpiWin) and MW = 226.32 g/mol
- Water solubility: Test substance was solubilized in DMSO to improve solubility
- Precipitation: observed at 5000 ug/plate
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity/range-finding test was carried out to determine the toxicity of the test material and to select the appropriate dose levels for use in the main test (See in "Remarks on results including tables and figures"). Cytotoxicity as based on a reduction in the background number of revertants per plate was observed at 5000 ug/plate for TA100 strain.


COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. The comparison was made with the historical control ranges for 2003 and 2004 of the corresponding Testing Laboratory.


ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material was non-toxic to the bacterial background lawns of the strains of bacteria used excepted at 5000 ug/plate for TA100 strain.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

1.) Preliminary Cytotoxicity Test:

The test material was toxic at 5000 ug/plate to the strain of Salmonella used (TA100). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The number of revertantcolonies for the toxicity assay were:

With (+) or without (-) Metabolic Activation (S9)

Strain

Dose (μg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

156

153

137

139

120

142

130

119

135

127

100SP

+

TA100

94

125

142

128

119

106

117

158

127

145

113SP

P: Precipitate S: sparse bacterial background

 

2.) Mutation Test:

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (See Tables of results in “Background attached material”) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number ofrevertantcolonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5 (See Tables of results in “Background attached material”).

In the first experiment, the test material caused a visible reduction in the bacterial background lawn to all of the tester strains except TA102, both with and without metabolic activation, at 5000 ug/plate. The toxic response of the test material to the tester strains was not as evident in

the second experiment however, with weakened lawns only observed in Salmonella strains TAl535 and TA1537. There was no toxicity observed to TA102 in either experiment. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. A fine, greasy precipitate was observed at 5000 ug/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation. All of the positive control chemicals used in the test induced marked increases in the frequency of

revertant colonies thus confirming the activity of the S9 -mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested, with any dose of the test material, either with or without metabolic activation. In consequence, the test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction. The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Reverse Mutation Study", Method Bl4 of Commission Directive 92/69/EEC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimurium strains TA1535, TA1537, TAl02, TA98 and TA100 were treated with the test material using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 15 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using a similar dose range to Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first experiment, the test material caused a visible reduction in the bacterial background lawn to all of the tester strains except TAl02, both with and without metabolic activation, at 5000 ug/plate. The toxic response of the test material to the tester strains was not as evident in the second experiment however, with weakened lawns only observed in Salmonella strains TA1535 and TA1537. There was no toxicity observed to TA102 in either experiment. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. A fine, greasy precipitate was observed at 5000 ug/plate, this did not prevent the scoring of revertant

colonies.

Conclusion. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.