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
Study period:
March 2007 - May 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
lower application volume
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
lower application volume
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
none

Method

Target gene:
Salmonella typhimurium LT2
Strains: TA 1535, TA 97a, TA 98, TA 100 and TA 102
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 98
Additional strain characteristics:
other: hisD3052, uvrB, pKM 101, rfa
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 metabolic activation
Species / strain:
other: 97a
Additional strain characteristics:
other: hisD6610, uvrB, pKM 101, rfa
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 metabolic activation
Species / strain:
S. typhimurium TA 100
Additional strain characteristics:
other: hisG46, uvrB, pKM 101, rfa
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 metabolic activation
Species / strain:
S. typhimurium TA 102
Additional strain characteristics:
other: hisG428, pKM 101, rfa
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 metabolic activation
Species / strain:
S. typhimurium TA 1535
Additional strain characteristics:
other: hisG46,
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 metabolic activation
Test concentrations with justification for top dose:
Experiment I (plate incorporation method): conc.: 625/187 162/19/6 µg/plate
Experiment II (pre-incubation method): conc.: 626 1 313 1157 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: [water]; The test item was dissolved in deionised water.
- Justification for choice of solvent/vehicle: test item was completely soluble in water, and water does not have any efects on the viability of the bacteria or the number of spontaneous revertants.
Controlsopen allclose all
Solvent controls:
yes
Remarks:
water
Positive controls:
yes
Remarks:
TA 97a, TA 98 and TA 102
Positive control substance:
other: 4-Nitro-1 ,2-phenylene diamine (80 µg/plate)
Remarks:
without metabolic activation
Solvent controls:
yes
Remarks:
water
Positive controls:
yes
Remarks:
TA 100, TA 1535
Positive control substance:
sodium azide
Remarks:
without metabolic activation

Migrated to IUCLID6: (6 µg/plate)
Solvent controls:
yes
Remarks:
water
Positive controls:
yes
Remarks:
TA 97a, TA 100, TA 102 and TA 1535
Positive control substance:
other: 2-Aminoanthracene (3 µg/plate)
Remarks:
with metabolic activation
Solvent controls:
yes
Remarks:
water
Positive controls:
yes
Remarks:
TA 98
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation

Migrated to IUCLID6: (40 µg/plate)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 12 h

PLATE INCORPORATION METHOD (first experiment):
the test solution of the appropriate concentration were membrane filtrated into sterile vessels.
Top agar basis was melted in a microwave oven, after melting, 10 mL of histidinebiotin-
solution 0.5 mMol per 100 mL basis was added and the bottle was placed in the water bath at 45 °C.
0.1 mL of the appropriate solution of the test item were given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix, 2 mL Top-Agar were added. The mixture
was gently vortexed, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula. The plates were c1osed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 °C.

PRE-INCUBATION METHOD (second experiment)
Per strain and dose, four plates with and four plates without S9 mix were used. 10 mL of
the test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidinebiotin- solution 0.5 mMol per 100 mL basis was added and the bottle was placed in the water bath at 45 °C.
0.1 mL of the appropriate solution of the test item were given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain, 0.5 mL phosphate buffer (only far treatments without S9) or 0.5 mL S9 mix were added. The mixture was incubated in an incubation chamber at 3rC for 20 minutes. During this time the vessels were aerated through ca refu I shaking. Then 2 mL top agar were added. The mixture was vortexed gently, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula. The plates were c1osed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 °C.


NUMBER OF REPLICATIONS:
Four replicates, with/without S9, for each solvent which was used in the test.

Evaluation criteria:
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
Statistics:
STATISTICAL METHDODS USED:
The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: First Experiment
Species / strain:
other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Second Experiment
Additional information on results:
OBSERVATION

First Expriment:
- The treatments for the confirmation of the genotype, the sterility control and the determination of the titre didn't show any inconsistencies
- The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory
- All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolie activation.
- No signs of toxicity towards the tested strains could be observed (The background lawn was visible and the number of revertant colonies was
not reduced)
- No significant increase of the number of revertant colonies in the treatments with and without metabolie activation could be observed.
-No concentration-related increase over the tested range was found.

Second Expriment:
- The treatments for the confirmation of the genotype, the sterility control and the determination of the titre didn't show any inconsisteneies
- The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory.
- All positive controls showed mutagenic effeets with and without metabolie activation.
- No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was
not significantly reduced.
- No signifieant inerease of the number of revertant eolonies in the treatments with and without metabolie activation was observed
- No coneentration-related inerease over the tested range was found.

COMPARISON WITH HISTORICAL CONTROL DATA:
The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory historical data of the laboratory (in the first and in the second experiment).

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Applicant's summary and conclusion

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

The test item is considered to ben non-mutagenic in the bacterial reverse mutation test.
Executive summary:

First Experiment:

Five concentrations of the test item, dissolved in tetrahydrofuran (THF) (ranging from nominally 625 to 6.25 µg/plate) were used. Five genetically manipulated strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolie activation system (S9) for 48 hours, using the plate incorporation method.

None of the concentrations caused an increase of the revertants in the tested strains. The test item didn't show any mutagenic effects in the first experiment. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Second Experiment:

To verify the results of the first experiment, a second experiment was performed, using three concentrations of the test item (ranging fram nominally 625 to 156.25 µg/plate)

and a modification in study performance (pre-incubation method). The test item didn't show mutagenic effects in the second experiment. No signs of toxicity towards the bacteria could be observed. The sterility contral and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative contrals were in the normal range. All positive contrals showed mutagenic effects with and without metabolic activation.