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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic activity of the test item was investigated in two fully reliable bacterial reverse mutation assays (Ames test; test strains used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 102), in an in vitro gene mutation study in mammalian cells (Chinese hamster V79 cells; HPRT test) and in one in vitro chromosome aberration test in human lymphocytes. Negative results were obtained in all tests with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

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

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

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

Target gene:

hypoxanthine guanine phosphoribosyl transferase Locus (HPRT)

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

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

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

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)

Reference 2

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 21, 2015 to June 12, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Guidelines followed

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine Gene

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

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

Untreated negative controls:

yes

Remarks:

Reverse osmosis Water

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl Sulfoxide

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-Aminoanthracene, 4-NOPD

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:

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 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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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

The spontaneous reversion rates in the negative control are in the range of in house historical data.

Conclusions:

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

It can be concluded that the test item Hostavin 3055 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 metaboloc 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.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 03, 2015 to October 21, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human Lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Absence and Presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 1.25 (T1), 2.5 (T2) and 5 (T3) mg/mL
Nc- negative control
VC - Vehicle control
T1- Test item concnetration 1
T2- Test item concnetration 2
T3- Test item concnetration 3

Untreated negative controls:

yes

Remarks:

Phosphate buffer solution

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl Sulfoxide

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Remarks: Without metabolic activation : EMS; Ethyl methanesulfonate ____________With metabolic activation: CPA; Cyclophosphamide Monohydrate

Key result

Species / strain:

lymphocytes: Human Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The maximum concentration 5 mg/mL was selected as the highest concentration for the treatment based on the cytotoxicity experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Human Lymphocytes

Remarks:

Migrated from field 'Test system'.

RESULTS

CYTOTOXIC EXPERIMENT

The cytotoxicity due to treatment with test item was assessed based on the mitotic index. The cytotoxicity experiment was conducted at the concentrations of 1.25 (T1), 2.5 (T2) and 5 (T3) mg/mL of culture media. A reduction in mitotic index was observed in the treated concentrations, both in the absence and in the presence of metabolic activation (1%), respectively.

In the absence of S9 mix, the mean mitotic index observed was 0.00 (T1), 0.00 (T2) and 0.00 (T3) and 10.10 (VC). In the presence of S9 mix, the mean mitotic index observed was 0.00 (T1), 0.00 (T2) and 0.00 (T3) and 9.39(VC).All these tested concentrationsviz.,1.25 (T1), 2.5 (T2) and 5 (T3) mg/mL of culture mediashowed cytotoxicity.

Hence further cytotoxicity experiment was conducted at the concentrations of 0.031 (T4), 0.063 (T5) and 0.125 (T6) mg/mL of culture media.In the absence of S9 mix, the mean mitotic index observed was 9.59 (T4), 7.50 (T5) and 0.25 (T6) and 10.10 (VC). In the presence of S9 mix, the mean mitotic index observed was 10.19 (T4), 7.38 (T5) and 0.25 (T6) and 9.39(VC).

In the second cytotoxicity experiment, the highest test concentration 0.125 (T6) mg/ mLof culture mediashowed 97% reduction the mitotic index when compared of the respective vehicle control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence this concentration was not selected for the main study.  

Hence, 0.063 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.

PHASE I

In the experiment, the cultures were exposed to HOSTAVIN 3055 for a short period of time (4 h) both in the absence and in the presence of metabolic activation system (1%). The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 1.000 (T3) and 9.000 (PC) in the absence of metabolic activation and 0.333 (NC), 0.667 (VC), 0.667 (T1), 0.667 (T2), 0.667 (T3) and 8.667 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.015 (T1), 0.031 (T2) and 0.063 (T3) mg HOSTAVIN 3055/mL and positive controls, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamideat the concentration of30 µg/mL in the presence of metabolic activation (1%) causedsignificant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.

Treatment with test item in the absence and presence of S9 mix, there was no reduction in mitotic index was observed at the tested concentrations. The observed mitotic index in the absence of metabolic activation were 10.04, 9.72, 9.15, 9.72 and 7.13 and in the presence of metabolic activation were 9.66, 9.87, 9.47, 9.68 and 7.52 for negative control, vehicle control, T1, T2 and T3 concentrations respectively.

PHASE II

The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the Phase II, test item treatment concentrations used were 0.015, 0.031 and 0.063 mg HOSTAVIN 3055/mL culture both in presence and in absence of metabolic activation (2%). The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation the duration of exposure was 24 hours. The mean percent aberrant cells were0.333(NC),0.333(VC),0.333(T1),0.667(T2), 1.000 (T3) and 9.667 (PC) in the of absence metabolic activation and0.667(NC),0.333(VC), 1.000 (T1),0.667(T2),0.667(T3) and 9.000 (PC) in the presence of metabolic activation at concentrations of 0.00 (NC), 0.00 (VC), 0.015 (T1), 0.031 (T2) and 0.063 (T3) mg HOSTAVIN 3055 /mL of culture and positive control, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamideat the concentration of30 µg/mL in the presence of metabolic activation (2%) causedsignificant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.

The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.

Treatment with test item in the absence and presence of S9 mix, there was no reduction in mitotic index was observed at the tested concentrations. The observed mitotic index in the absence of metabolic activation were 10.50, 9.43, 9.35, 8.82 and 7.39 and in the presence of metabolic activation were 9.11, 9.44, 9.51, 9.22 and 7.44 for negative control, vehicle control, T1, T2 and T3 concentrations respectively.

Conclusions:

In conclusion, under the experimental conditions and results of this study, it is concluded that HOSTAVIN 3055 is “non-mutagenic” both in the presence (1% and 2%) and in the absence of metabolic activation in this assay.

Executive summary:

SUMMARY

This study was conducted to determine the chromosomal aberration induction potential of HOSTAVIN 3055 in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on26September 2014.

The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 68 h mitogenic stimulation. The cells were treated with metaphase arresting substance (colcemid) 4 h prior to harvesting and stained. The metaphase cells were analysed microscopically and a minimum number of 1000 cells per slide were counted and number of metaphases were recorded in different fields to determine the mitotic index. The number of aberrant cells with aberrations were recorded (150 cells per slide) to calculate percent aberrant cells.

On the basis of solubility and precipitation test, Dimethyl Sulphoxide (DMSO) was selected as the vehicle for treatment. The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37 ± 2 °C. Significant change in pH was not observed at 0 h and 4 h when compared with negative controls. There was no precipitation observed at 5 mg/mL concentration. Hence 5 mg/mL was selected as the highest concentration for cytotoxicity test.

CYTOTOXIC EXPERIMENT

Before conducting the chromosomal aberration study, HOSTAVIN 3055 was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 1.25 (T1), 2.5 (T2) and 5 (T3) mg/mL of culture media. Cytotoxicity was observed in the treated concentrations both, in the absence and in the presence of metabolic activation (1%). 

In the absence of S9 mix, the mean mitotic index observed was 0.00 (T1), 0.00 (T2) and 0.00 (T3) and 10.10 (VC). In the presence of S9 mix, the mean mitotic index observed was 0.00 (T1), 0.00 (T2) and 0.00 (T3) and 9.39(VC).All these tested concentrationsviz.,1.25 (T1), 2.5 (T2) and 5 (T3) mg/mL of culture mediashowed cytotoxicity.

Hence further cytotoxicity experiment was conducted at the concentrations of 0.031 (T4), 0.063 (T5) and 0.125 (T6) mg/mL of culture media.In the absence of S9 mix, the mean mitotic index observed was 9.59 (T4), 7.50 (T5) and 0.25 (T6) and 10.10 (VC). In the presence of S9 mix, the mean mitotic index observed was 10.19 (T4), 7.38 (T5) and 0.25 (T6) and 9.39(VC).

In the second cytotoxicity experiment, the highest test concentration 0.125 (T6) mg/ mLof culture mediashowed around 97% reduction the mitotic index when compared of the respective vehicle control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence this concentration was not selected for the main study.  

Hence, 0.063 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.

The main study was performed in twoindependentphases;

MAIN STUDY

PHASE I

In the experiment, the cultures were exposed to HOSTAVIN 3055 for a short period of time (4 h) both in the absence and in the presence of metabolic activation system (1%). The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 1.000 (T3) and 9.000 (PC) in the absence of metabolic activation and 0.333 (NC), 0.667 (VC), 0.667 (T1), 0.667 (T2), 0.667 (T3) and 8.667 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.015 (T1), 0.031 (T2) and 0.063 (T3) mg HOSTAVIN 3055/mL and positive controls, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamideat the concentration of30 µg/mL in the presence of metabolic activation (1%) causedsignificant increase in percent aberrant cells.Even though the analysis did not reveal any statistical significance, the increase was biologically significant.

Treatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index was observed at the tested concentrations. The observed mitotic indexin the absence of metabolic activation were 10.04, 9.72, 9.15, 9.72 and 7.13 andin the presence ofmetabolic activation were 9.66, 9.87, 9.47, 9.68 and 7.52 for negative control, vehicle control, T1, T2 and T3 concentrations respectively.

Note: NC: Negative control; VC: Vehicle control; T1: Test concentration1; T2: Test concentration 2; T3: Test concentration 3; PC: Positive Control.

PHASE II

The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the Phase II, test item concentrations used were 0.015, 0.031 and 0.063 mg HOSTAVIN 3055/mL culture both in presence and in absence of metabolic activation (2%). The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation the duration of exposure was 24 hours. The mean percent aberrant cells were0.333(NC),0.333(VC),0.333(T1),0.667(T2), 1.000 (T3) and 9.667 (PC) in the of absence metabolic activation and0.667(NC),0.333(VC), 1.000 (T1),0.667(T2),0.667(T3) and 9.000 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.015 (T1), 0.031 (T2) and 0.063 (T3) mg HOSTAVIN 3055 /mL of culture and positive control, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamideat the concentration of30 µg/mL in the presence of metabolic activation (2%) causedsignificant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.

The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.

Treatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index was observed at the tested concentrations. The observed mitotic indexin the absence of metabolic activation were 10.50, 9.43, 9.35, 8.82 and 7.39 andin the presence ofmetabolic activation were 9.11, 9.44, 9.51, 9.22 and 7.44 for negative control, vehicle control, T1, T2 and T3 concentrations respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

not mandatory

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species specific effects of the substance. Therefore the results of the in vitro data are regarded as relevant for humans.

Additional information

Mutagenicity of the submission substance has been investigated in vitro in two reliable bacterial reverse mutations assays, which both revealed negative results in the presence and the absence of metabolic activation in all strains tested. These data are supported by another bacterial reverse mutation assay of lower reliability.

Negative results were also obtained in a genotoxicity assay and a cytogenicity assay in mammalian cells in vitro (HPRT test and chromosome aberration test, both in the presence and the absence of metabolic activation).

Hostavin 3055 does not have to be classified for mutagenicity since this substance did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation, in thein vitrogene mutation assay or in thein vitrochromosome aberration test.

Justification for classification or non-classification

Due to the consistent negative findings in all in vitro assays no classification for mutagenicity is recommended according to the criteria of Regulation (EC) No 1272/2008.