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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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:
Batch: PR-001 GDF97053
Physical description: amber solid.
Storage: Room temperature

Method

Target gene:
Histidine for Salmonella typhimurium
Tryptophan for 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):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat livers (S9 fraction)
Test concentrations with justification for top dose:
Rangefinding test:
6.67 - 5000 μg/plate

Concentration range in main test:
With metabolic activation: 10 - 5000 μg/plate (10.0, 33.3, 100, 333, 1000, 5000 μg/plate)
Without metabolic activation: 10 - 5000 μg/plate (10.0, 33.3, 100, 333, 1000, 5000 μg/plate)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See remarks.
Remarks:
Used with S9 mix: benzo(a)pyrene, 2-aminoanthracene. Used without S9 mix: 2-nitrofluorene, sodium azide, ICR-191, 4-nitroquinoline-N-oxide.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: overnight.
- Exposure duration: 52 hrs (+/- 4 hrs)
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 52 hrs (+/- 4 hrs)


SELECTION AGENT (mutation assays): Not applicable.


NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method:
Bacterial Background Lawn Evaluation -
The condition of the bacterial background lawn was evaluated for evidence of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level.

Counting revertant colonies -
The number of revertant colonies per plate for the vehicle controls and all plates containing test article were counted manually. The number of revertant colonies per plate for the positive controls were counted by automated colony counter.


OTHER EXAMINATIONS: None
Evaluation criteria:
Criteria For A Positive Response:

Once the criteria for a valid assay had been met, responses observed in the assay were evaluated as follows:

1. Tester Strains TA98, TA100 and WP2uvrA
For a test article to be considered positive, it had to produce at least a 2-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.

2. 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.

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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation seen at dose level of 1000 μg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
No cytotoxicity was observed in either the presence or absence of S9 mix as evidenced by no decrease in the number of revertants per
plate. The bacterial background lawns were evaluated as normal up to the 1,000 μg per plate dose. Lawns above this dose were obscured by precipitate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test substance did not cause a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absense of microsomal enzymes prepared from Aroclor 1254 induced rat liver (S9).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The test substance did not cause a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor 1254 induced rat liver (S9) microsomal fraction. The positive controls demonstrated the sensitivity of the test and the negative controls were within historical limits.

Metabolic activation

Test Substance Concentration (μg/plate) Resulting in:

Cytotoxicity in Preliminary Test

Cytotoxicity in Main Test

Precipiatation

Genotoxic Effect

Present

 

 

 

 

Test 1

>5000

>5000

1000

-

Test 2

>5000

>5000

1000

-

Absent

 

 

 

 

Test 1

>5000

>5000

1000

-

Test 2

>5000

>5000

1000

-

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The notified chemical was not mutagenic to bacteria under the conditions of the test.
Executive summary:

Introduction:

The test substances mutagenic activity was investigated in a Salmonella - Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay. This assay evaluated the test article and/or its metabolites for their ability to induce reverse mutations at the histidine locus in the genome of specific Salmonella typhimurium tester strains and at the tryptophan locus in an Escherichia coli tester strain, both in the presence and absence of an exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclor-induced rat liver (S9).

Method:

The doses tested in the mutagenicity assay were selected based on the results of a dose range finding study using tester strains TA100 and WP2uvrA and ten doses of test article ranging from 5,000 to 6.67 μg per plate, one plate per dose, both in the presence and absence of S9 mix. The tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1 535, TA1537, and Escherichia coli tester strain WP2MvrA. The assay was conducted with six doses of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The doses tested were 5,000, 1,000, 333, 100, 33.3, and 10.0 μg per plate in both the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in an independent experiment. Conclusions:

The test substance did not cause a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor 1254 induced rat liver (S9)