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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Details on test material:
- Name of test material (as cited in study report): 852 IMIDE ( = tetrahydrocyclopenta[c]pyrrole-1,3(2H,3aH)-dione)

- Physical state: flakes

- Storage condition of test material: stable under normal conditions of storage

Method

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:
S-9 mix (microsomial fraction of Sprague-Dawley rat liver)
Test concentrations with justification for top dose:
The range of concentrations to be studied has been defined after a preliminary assay of cytotoxicity on the TA 100 strain.
the final test concentrations are : 500, 1500, 500, 150 and 50 µg of the test substance/plate.
Vehicle / solvent:
Methanol.
A mother solution of the test item has been prepared, at 166.66 mg/ml, in methanol.
The maximal volume of methanol added per plate is 30 µl.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
B - propiolactone ; 2-anthramine
Remarks:
Controls of sterility of prepared solutions and of S9mix have been realized.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 1 hour at 37°C for the metabolic acivation assays
- Exposure duration: 48 h at 37°C (incubation period)
- tests performed with and without metabolic activity on all strains.

DETERMINATION OF CYTOTOXICITY
- counting of revertant colonies


Evaluation criteria:
the result of the experimentation is considered negative if the rate of revertant remains :
- inferior to the triple of rate of spontaneous reversions, for strains TA 1535 and TA1537 (with and without metabolic activation)
and
- inferior to the double of rate of spontaneous reversions for strains TA98, TA 100 and E. Coli WP2 (with and without metabolic activation).
Statistics:
comparison to historical experimental data of the LEMI laboratory :
- for salmonella typhimurium : data from 25/06/94 to 01/12/2002.
- for escherichia coli : data from 01/04/98 to 01/12/2002.

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
Untreated negative controls validity:
valid
Positive controls validity:
valid
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

No increase of revertants number has been shown during this study, for all the concentrations tested (5000, 1500, 500, 150 and 50 µg/plate), with or without metabolic activation, for TA 1535, 1537, 98 and 100 strains and for E. Coli WP2 strain.