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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:
January 2008 - May 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study: Planning and conduct of the study were based on the OECD guideline "Genetic Toxicology: Salmonella typhimurium, Reverse Mutation Assay", OECD No. 471 OECD Guidelines for the Testing of Chemicals „Bacterial Reverse Mutation Test“ Part 471, adopted July 21st, 1997 EU-Guideline 67/548 EWG, last changed by 29th amendment, Annex V, Method B.13/14 “Mutagenicity – Salmonella typhimurium, reverse mutation assay”, adopted May 19th, 2000

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD 471
Deviations:
no
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

Method

Target gene:
his- of Salmonella typhimurium LT2
Strains: TA 1535, TA 97a, TA 98, TA 100 and TA 102
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: hisD3052, uvrB, pKM 101, rfa
Species / strain / cell type:
other: TA97a
Additional strain / cell type characteristics:
other: hisD6610, uvrB, pKM 101, rfa
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: hisG46, uvrB, pKM 101, rfa
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: hisG428, pKM 101, rfa
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type 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.: 5047 / 1514 / 505 / 151 / 51 µg/plate
Experiment II (pre-incubation method): conc.: 5031 / 2516 / 1258 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [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
Negative solvent / vehicle 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 system
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Remarks:
TA 100 and TA 1535
Positive control substance:
sodium azide
Remarks:
without metabolic activation system

Migrated to IUCLID6: (6 µg/plate)
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Remarks:
TA 98
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation system

Migrated to IUCLID6: (40 µg/plate)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
yes
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 system
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Preincubation period: 12 hours.
PLATE INCORPORATION METHOD (First 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 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 closed, 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 for treatments without S9) or 0.5 mL S9 mix were added. The mixture was incubated in an incubation chamber at 37°C for 20 minutes. During this time the vessels were aerated through careful 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 closed, 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
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
Metabolic activation:
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:
OBSERVATION

FIRST EXPERIMENT:
• The treatments for the confirmation of the genotype, the sterility control and the determination of the titer did not show any inconsistencies.
• All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation.
• No signs of toxicity towards the tested strains could be observed:
• The background lawn was visible; the number of revertant colonies was not significantly reduced.
• No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed.
• No concentration-related increase over the tested range was found.

SECOND EXPERIMENT:
• The treatments for the confirmation of the genotype, the sterility control and the determination of the titer did not show any inconsistencies.
• All positive controls showed mutagenic effects with and without metabolic activation.
• No signs of toxicity towards the tested strains could be observed:
• The background lawn was visible; the number of revertant colonies was not significantly reduced.
• No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed.
• No concentration-related increase 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).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: First Experiment

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 deionised water (ranging from 5047 to 51 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 metabolic activation system (S9) for 48 hours, using the plate incorporation method.

None of the concentrations caused an increase in the number of revertant colonies in the tested strains.

The test item didn't show any mutagenic effects in the first experiment. The test item didn't show any cytotoxicity towards the bacteria.

The sterility control and the determination of the titer did not 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 from 5031 to 1258 µg/plate) and a modification in a study performance (pre-incubation method).

The test item didn’t show any mutagenic effects in the second experiment, either. No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titer did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive showed mutagenic effects with and without metabolic activation.

Under the conditions of the test, the test item did not show mutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined.

The test item is considered as "not mutagenic” under the conditions of the test.