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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:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline comfirmed study

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his, trp
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa wall mutation, deletion of the uvrB gene, contain the R-factor plasmid, pKM101
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: uvrA DNA repair deficiency
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from aroclor induced rat liver
Test concentrations with justification for top dose:
The doses tested were 33.3,100, 333, 1000,3330, and 5000 ug per plate in both the presence and absence of S9 mix.
Vehicle / solvent:
- Vehicle: ethanol
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
cells and medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
benzo(a)pyrene
Remarks:
TA98
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA100, 1535, 1537, WP2MvrA
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
2-nitrofluorene
Remarks:
TA98
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
TA100, TA1535
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: ICR-191
Remarks:
TA1537
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
WP2MvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation

DURATION
When S9 mix was not required, 100 /µL of tester strain and 100 /µL of vehicle or test article dose were added to 2.5 mL of molten selective top agar (maintained at 45 ± 2°C). When S9 mix was required, 500 /µL of S9 mix, 100 /uL of tester strain and 100 /µL of vehicle or test article dose were added to 2.0 mL of molten selective top agar. After the required components had been added, the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay had solidified, the plates were inverted and incubated for 52 ± 4 hours at 37 ± 2°C. Positive control articles were plated using a 50 /µL plating aliquot.

NUMBER OF REPLICATIONS: in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Dose Rangefinding Assay
The growth inhibitory effect (cytotoxicity) of the test article to the test system was determined in order to allow the selection of appropriate doses to be tested in the mutagenicity assay.

Design
The dose rangefinding assay was performed using tester strains TAIOO and WP2MvrA both in the presence and absence of S9 mix. Ten doses of test article were tested at one plate per dose. The test article was checked for cytotoxicity up to a maximum concentration of 5 mg per plate.
Evaluation criteria:
Assay Evaluation Criteria
Once the criteria for a valid assay had been met, responses observed in the assay were evaluated
as follows:
Tester Strains TA98, TA100, and WP2uvrA. For a test article to be considered positive, it had to produce at least a 2-foId increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
Tester Strains TA1535 and TA1537. For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
Statistics:
For all replicate platings, the mean revertants per plate and the standard deviation were calculated. The results of these calculations are presented in tabular form in the Data Tables section of this report. The historical control data are presented after the data tables.

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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
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
Additional information on results:

RANGE-FINDING/SCREENING STUDIES: see above in study design

COMPARISON WITH HISTORICAL CONTROL DATA: yes, data were presented after data tables

ADDITIONAL INFORMATION ON CYTOTOXICITY: no
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 metabolic activation
negative without metabolic activation

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test substance did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver (S9)
Executive summary:

The results of the dose rangefinding study were used to select the doses tested in the mutagenicity assay. The doses tested were 33.3, 100, 333,1000, 3330, and 5000 /µg per plate in both the presence and absence of S9 mix. In the initial mutagenicity assay, all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains either in the presence or absence of S9 mix. In the confirmatory assay, all data generated with tester strain TA98 in the presence of S9 mix, and with tester strains TA100, TA1535, TA1537, and WP2uvrA in both the presence and absence of S9 mix, were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of these tester strain/activation condition combinations. In this experiment, the mean positive control value for tester strain TA98 in the absence of S9 mix was not acceptable (did not exhibit at least a 3-fold increase over the mean vehicle control value). For this reason, the test article-treated plates were not scored and tester strain TA98 was retested in the absence of S9 mix in Experiment 21285-Dl. In Experiment 21221-Dl, the mean positive control value for tester strain TA98 in the absence of S9 mix was lower than routinely observed in this laboratory, although it did exhibit at least a 3-fold increase over the mean vehicle control for this tester strain. For this reason, tester strain TA98 was retested in the absence of S9 mix in Experiment 21221-D2. In Experiment 21221-D2, all data generated with tester strain TA98 in the absence of S9 mix were acceptable, and no positive increases in the mean number of revertants per plate were observed. All criteria for a valid study were met.