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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:
gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 21, 2015 to June 12, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report):Hostavin 3055
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type:
- Physical state:Liquid / light yellowish
- Analytical purity:98.3% (w/w)
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.:DEF2101115
- Expiration date of the lot/batch:July 07, 2020
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions:Stable at room temperature
- Storage condition of test material:Room Temperature (20 to 30°C)
- Other:

Method

Target gene:
Histidine Gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
nitroreductase deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.0002, 0.001, 0.002, 0.005 and 0.016 mg/plate both in absence (-S9) and in the presence (+S9) of metabolic activation
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Remarks:
Reverse osmosis water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
other: 2-Aminoanthrancene, 4-NOPD

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium 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
Key result
Species / strain:
S. typhimurium TA 1537
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

Any other information on results incl. tables

Additional information on results

                                                         Solubility Record

Solvent used

RO (reverse osmosis) Water

Dimethyl sulfoxide

Quantity of test item

50 mg

50 mg

Volume of vehicle added

1000 µL

1000 µL

Final Concentration

50 mg /mL

50 mg /mL

Solubility status

Insoluble

Soluble

As mentioned in the above table, solubility of test item was checked in RO water and found insoluble. So the solubility was checked in Dimethyl sulfoxide (DMSO). The test item was found soluble in DMSO at 50 mg/ml to give final treatment concentration of 5 mg/plate (recommended maximum test concentration for soluble non-cytotoxicity substances). Therefore, DMSO was chosen as solvent for the study.

Precipitation

Precipitation was checked as insolubility to assess precipitation in the final mixture under the actual test conditions and evident to the unaided eye. Test item dissolved in DMSO at 50 mg/mL concentration was checked for precipitation. Details are given in table below:

Precipitation Record

Overlay agar volume

Test item preparation volume

Concentration/Plate

Result

2 mL

100 µL

5 mg

No Precipitation

An amount of test item preparation (50 mg/mL) was added 2 ml of overlay agar (Top agar) in test tubes to give test item concentration of 5 mg/plate and plated on minimal glucose agar (MGA) plates. Precipitation was not observed. Therefore, 5 mg/plate was selected as the highest concentration for pre-experiment.

Range-Finding/Screening studies

The pre-experiment was performed with TA 100 and TA 98 strain ofSalmonella typhimuriumand with eight different concentrations of the test item prepared with half log intervals and three plates were used for each dose level and for the controls. 5 mg/plate were selected as the highest dose in the pre-experiment basedon the solubility and precipitation test. Following doses were selected for the pre-experiment i.e., 0.001, 0.002, 0.005, 0.016, 0.050, 0.501, 1.582 and 5 mg/plate.

The results were evaluated based on the reduction of revertant colony count and bacterial background lawn.

Toxicity was observed in the tested concentrations 5 (T8) and 1.582 (T7) mg/plate. Reduction in colony count and microcolonies were observed in the tested concentrations 0.501 (T6) and 0.050 (T5) mg/plate both in absence and in the presence of metabolic activation. At treated concentrations 0.016 (T4) to 0.001 (T8) mg/plate, no reduction in colony count and background lawn were observed both in absence and in the presence of metabolic activation, when compared to that of the negative control group.

Based on the results of pre-experiment, following doses were selected for the main study trials: 0.0002, 0.001, 0.002, 0.005 and 0.016 mg/plate both in absence (-S9) and in the presence (+S9) of metabolic activation.

Comparision with Historical Control Data

Applicant's summary and conclusion

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

It can be concluded that the test item did not induce gene mutations either by base pair substitution or by frame shifts in the genome of the strains used under the conditions of the assay with and without metabolic activation.
Executive summary:

Summary

This study was performed to assess the mutagenic potential of Hostavin 3055 to induce gene mutations in comparison to vehicle (solvent) control according to the plate incorporation method (Trial-I) and the pre-incubation method (Trial-II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicates. The test item was tested at the following concentrations i.e., 0.0002, 0.001, 0.002, 0.005 and 0.016 mg/plate both in presence (+S9) and in absence (-S9) of metabolic activation(The test item concentration values have been incorporated upto three decimal places in this report).

No significant increase in revertant colony numbers in any of the tester strains were observed following treatment with Hostavin 3055 at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The spontaneous reversion rates in the negative, vehicle and positive controls were within the range of in-house historical data.

The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.