3-dodecyl-1-(2,2,6,6-tetramethyl-4-piperidyl)pyrrolidine-2,5-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 19, 2016 to April 07, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-dodecyl-1-(2,2,6,6-tetramethyl-4-piperidyl)pyrrolidine-2,5-dione

EC Number:

279-242-6

EC Name:

3-dodecyl-1-(2,2,6,6-tetramethyl-4-piperidyl)pyrrolidine-2,5-dione

Cas Number:

79720-19-7

Molecular formula:

C25H46N2O2

IUPAC Name:

3-dodecyl-1-(2,2,6,6-tetramethylpiperidin-4-yl)pyrrolidine-2,5-dione

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Clariant India Limited and DEF2101115
- Expiration date of the lot/batch:
July 07, 2020
- Purity :98.3% (w/w)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Stable at room temperature
- Stability under test conditions:Unknown
- Solubility and stability of the test substance in the solvent/vehicle:
Solubile in DMSO

Method

Target gene:

hypoxanthine guanine phosphoribosyl transferase Locus (HPRT)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:National Centre for Cell Science, Pune, INDIA
- Suitability of cells:
- doubling time : 13-16 hours in stock cultures

- Methods for maintenance in cell culture if applicable:
Prior to mutagenicity testing the amount of spontaneous mutants were depressed by growing the cells for three days in HAT medium
The incubation of the cells in HAT-medium was followed by a recovery period of 24 hours in HT medium.
After this, these cells were returned to normal DMEM medium (complete culture medium) to grow the cells for three days to produce sufficient cell numbers
The cell lines from the stocks of the cleansed V79 cell line were cryopreserved as stock cultures in cryovials with freezing media (DMEM20 + 10% v/v DMSO) in liquid nitrogen

- Modal number of chromosomes:22 (±3)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
DMEM, 5%
- Properly maintained: [yes/no]
YES
- Periodically checked for Mycoplasma contamination: [yes/no]
Yes
- Periodically 'cleansed' against high spontaneous background: [yes/no]
Yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

AroclorTM 1254 induced liver homogenate (S9)

Test concentrations with justification for top dose:

62.5 µg/mL ,31.25 µg/mL ,15.63 µg/mL ,7.81 µg/mL both in the presence and absence of metabolic activation

highest cocentration - 62.5 µg/mL selected Since during cytotoxicity study 125μg/ml which shows 3.95% (-S9), and 1.66% (+S9) of Relative Survival indicating cytotoxicity, the RS of the other tested concentrations were found to be more than 50%

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO;
- Justification for choice of solvent/vehicle:
The test item was found soluble in DMSO. Therefore, DMSO was selected as solvent for the study

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; DMEM
- Cell density at seeding (if applicable):1.5x106 (single culture) and 5x102 (in duplicate) were seeded in DMEM10 for determination of mutation rate and toxicity, respectively

DURATION
- Exposure duration:4 hours
- Expression time (cells in growth medium):7days
- Selection time (if incubation with a selection agent):7days

SELECTION AGENT (mutation assays):6-thioguanine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;

OTHER EXAMINATIONS:
Mutant frequency

Evaluation criteria:

A test item is classified as positive if
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
• The increase is concentration-related
Any of the results are outside the distribution of the historical negative control data

Statistics:

Mann-Whitney test

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

during cytotoxicity experiment 125μg/ml which shows Relative Survival indicating cytotoxicity, the RS of the other tested concentrations were found to be more than 50% hence for main study 62.5 µg/mL was slected as highest cocentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In conclusion it is stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the HPRT locus in V79 cells of the Chinese hamster in the absence and presence of metabolic activation.
Therefore, HOSTAVIN 3055 were considered to be “ non-mutagenic” in this HPRT assay.

Executive summary:

This study was conducted to investigate the potential ofHostavin 3055to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The methods followed were as per OECD guideline No. 476, adopted on28^thJuly 2015.

The assay was performed in two independent experiment, using two parallel cultures each. The experiment I was performed with and without liver microsomal activation at a treatment period of 4 hours. Experiment II was performed for a treatment period of 4 hours with and 24 hours without metabolic activation.

125 µg/mL concentration was chosen as the highest dose for the cytotoxicity experiment, based on the solubility and precipitation properties of the test item.

The following concentrations were selected for the main study based on cytotoxicity results.

62.5 µg/mL ,31.25 µg/mL ,15.63 µg/mL ,7.81 µg/mL both in the presence and absence of metabolic activation

No relevant cytotoxic effect was observed as indicated by the Relative survival(RS) i.e., cloning efficiency (CE) of cells plated immediately after treatment, adjusted by any loss of cells during treatment, based on cell count, as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100%) with the RS for the test item. With exception of highest concentration i.e.,125μg/ml which shows 3.95% (-S9), and 1.66% (+S9) of Relative Survival indicating cytotoxicity, the RS of the other tested concentrations were found to be more than 50% and hence the same doses were selected for the main experiment (Phase-I and Phase-II).

PHASE-I

In culture I, the number of mutant colonies of NC, VC, T1 ,T2 ,T3 ,T4 and PC (EMS) were 5.3, 7.0, 7.7, 6.5, 11.1, 11.1, and 132.1/10⁶cells respectively in the absence of metabolic activation, number of mutant colonies of NC, VC, T1 ,T2 ,T3 ,T4 and PC (DMBA) were 11.8, 13.2, 14.2, 16.2, 21.9, 30.5 and 967.6 per 10⁶cells in presence of metabolic activation.

In culture II, the number of mutant colonies of NC, VC, T1 ,T2 ,T3 ,T4 and PC (EMS) were 5.1, 5.0, 7.8, 10.6, 10.9, 13.3 and 147.0/10⁶cells respectively and in the absence of metabolic activation, number of mutant colonies of NC, VC, T1 ,T2 ,T3 ,T4 and PC (DMBA) were 11.9, 16.3, 13.1, 19.2, 25.9, 27.8, and 874.2 per 10⁶cells in presence of metabolic activation.

PHASE-II

In culture I, the number of mutant colonies of NC, VC, T1 ,T2 ,T3 ,T4 and PC (EMS) were 6.4, 8.1, 11.1, 13.3, 14.2, 13.7, and 170.1/10⁶cells respectively in the absence of metabolic activation, number of mutant colonies of negative control, vehicle control and was T1 ,T2 ,T3 ,T4 and positive control (DMBA) were 11.3, 13.8, 15.3, 17.7, 19.3, 21.2 and 1160.3/10⁶cells in presence of metabolic activation.

In culture II, the number of mutant colonies of NC, VC, T1 ,T2 ,T3 ,T4 and PC (EMS) was 6.8, 9.0, 10.2, 11.4, 13.5, 15.7 and 175.9/10⁶cells respectively and in the absence of metabolic activation, number of mutant colonies of NC, VC, T1 ,T2 ,T3 ,T4 and PC (DMBA) was 10.6, 13.8, 15.5, 18.8, 19.5, 22.1 and 1092.8 per 10⁶cells respectively in presence of metabolic activation.

In both the cultures, there was no distinct increase in the mutant frequency ofHostavin 3055when compared to respective vehicle control and the induction factor does not exceed more than three times the corresponding vehicle controls.No significant and reproducible dose dependent increase in mutant colony numbers was observed in either the Phase I or Phase II of the experiment.

The positive controls used, EMS in the absence of metabolic activation andDMBAin the presence of metabolic activation, revealed a significant increase in mutant colonies and the induction factor is more than three times of vehicle control indicating that the test system was sensitive and the results are valid.

In the main experimentHostavin 3055does not show a distinct increase of the number of mutant colonies and thus proved the validity of test system and activity of the S9 mix.

Note:NC: Negative control; VC: Vehicle control; T1: Test concentration1; T2: Test concentration 2; T3: Test concentration 3; T4: Test concentration 4; PC: Positive Control;EMS: (ethyl methanesulfonate);DMBA:(Dimethyl benz(a)anthracene)