1-acetyl-4-(3-dodecyl-2,5-dioxo-1-pyrrolidinyl)-2,2,6,6-tetramethylpiperidine

Genetic toxicity: in vitro

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

Test material information

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.