Reaction mass of Fatty acids, tallow, Guerbet reaction Products, Ca salts and Fatty acids, tallow, Guerbet reaction Products, Na salts and Fatty acids, tallow, Guerbet reaction Products

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic activity of Licowax R21 S was investigated in one bacterial reverse mutation assay (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) and one in vitr ochromosome aberration study in human lymphocytes. Negative results were obtained in all performed genotoxicity tests.

Link to relevant study records

Reference

Reference 1

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:

September 30, 2015 to January 22, 2016

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0.0050, 0.0158, 0.0501, 0.1582 and 0.5 mg/plate both with and without metabolic activation

Untreated negative controls:

yes

Remarks:

RO water

Negative solvent / vehicle controls:

yes

Remarks:

Ethyl Alcohol

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine -4-NOPD, 2-Aminoanthracene, 2-AA

Details on test system and experimental conditions:

please refer to "Any other information on materials and methods"

Rationale for test conditions:

Conditions were chosen based on the results of the pre test.

Evaluation criteria:

A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the htreshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of solvent control such an increase is not considered biologically relevant.

Statistics:

none

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

test item Licowax R 21 S FL 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

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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 AND PRECIPITATION CHECK

To select an appropriate solvent and dose concentration range of the test item to be tested in pre-experiment, solubility and precipitation test were performed.Summary of solubility and precipitation check is as follows:

Solubility Record
Solvent used	RO (reverse osmosis) Water	Dimethyl sulfoxide	Acetone	Ethyl alcohol
Quantity of test item	50 mg	50 mg	50 mg	50 mg
Volume of vehicle added	1mL	1mL	1mL	1mL
Final Concentration	50 mg /mL	50 mg /mL	50 mg /mL	50 mg /mL
Solubility status	Insoluble	Insoluble	Insoluble	Soluble

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

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 inEthyl alcoholat 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	Precipitation
2 mL	75 µL	3.75 mg	Precipitation
2 mL	50 µL	2.5 mg	Precipitation
2 mL	25 µL	1.25 mg	Precipitation
2 mL	12.5 µL	0.625 mg	Precipitation
2 mL	10 µL	0.5 mg	Slight Precipitation

Different amounts of formulated test item (50 mg/ml) were added to overlay agar (top agar) in test tubes to give various test item concentrations (maximum 5 mg/plate) and plated on minimal glucose agar (MGA) plates. Precipitation was noticed at 5 mg/plate, 3.75 mg/plate, 2.5 mg/plate, 1.25mg/plate and 0.625mg/plate concentration which were assumed to interfere with the scoring. At treatment concentration 0.5 mg/plate slight precipitation was observed which was assumed non-interfering with the scoring. Therefore 0.5 mg/plate selected as highest concentration for pre-experiment

Range finding/Screening

In the pre-experiment, the concentration range of the test item was 0.0002 to 0.5 mg/plate based on the solubility and precipitation test.

No reduction in colony count as well as background lawn in any of the following concentrations tested; 0.0002, 0.0005, 0.0016, 0.0050, 0.0158, 0.0501, 0.1582 and 0.5 mg/plate both in absence and in the presence of metabolic activation, when compared to that of the vehicle control group.

Based on the results of pre-experiment, following doses were selected for the main study trials: 0.0050, 0.0158, 0.0501, 0.1582 and 0.5 mg/plate, both in absence (-S9) and in the presence (+S9) of metabolic activation

Conclusions:

It is concluded that the test item Licowax R 21 S FL 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 Licowax R 21 S FL 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 the Salmonella typhimurium strains 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., 0.0050, 0.0158, 0.0501, 0.1582 and 0.5 mg/plate, both in presence (+S9) and in absence (-S9) of metabolic activation.

No significant increase in revertant colony numbers in any of the tester strains were observed following treatment with Licowax R 21 S FL 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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 21, 2015 to July 27, 2016

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:

other: In vitro Mammalian Chromosome Aberration Test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:CLARIANT
- Expiration date of the lot/batch: November 13, 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Room Temperature (20 to 30°C)
- Stability under test conditions:Unknown
- Solubility and stability of the test substance in the solvent/vehicle:Soluble in Ethyl alcohol

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Adult healthy human beings
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Modal number of chromosomes:46

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI /5% Co2
- Properly maintained: [yes/no] YES

Metabolic activation:

with and without

Metabolic activation system:

mammalian microsome enzyme activation mixture (S9)

Test concentrations with justification for top dose:

Based on the results of cyto-toxicity experiment following doses were selected for the main study:
0.125, 0.25 and 0.5 mg/mL culture media

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol;
- Justification for choice of solvent/vehicle: test item found soluble in ethanol;

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;RPMI

DURATION
- Exposure duration: 4 hours

SPINDLE INHIBITOR (cytogenetic assays):colcemid

STAIN (for cytogenetic assays):Giemsa stain

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least one slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. All slides, including those of positive and negative controls, were independently coded before microscopic analysis. Scorer read only those slides, which were coded

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

Rationale for test conditions:

Test conditions were chosen based on results of pre experiment.

Evaluation criteria:

A test item can be classified as non-mutagenic if:
 The number of induced structural chromosome aberrations in all evaluated dose groups is within the range of the negative or vehicle control.
A test item can be classified as mutagenic if:
 The number of induced structural chromosome aberrations were increased at least 2-3 fold when compared to negative or vehicle control data.
And
 either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria are valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is 2-3 fold more when compared to the vehicle and negative control data.

Statistics:

Statistical significance was confirmed by means of the non-parametric Mann Whitney Test. However, both biological and statistical significance considered together.

Key result

Species / strain:

lymphocytes: human

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

This study was conducted to determine the chromosomal aberration induction potential of Licowax R 21 S FL 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, Ethyl alcohol 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 0.5 mg/mL concentration. Hence 0.5 mg/mL was selected as the highest concentration for cytotoxicity test.

1.1                 

Before conducting the chromosomal aberration study, Licowax R 21 S FL was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.125 (T1), 0.25 (T2) and 0.5 (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 10.49 (NC), 9.78 (VC), 9.44 (T1), 9.52 (T2), 9.58 (T3) and 9.21 (PC). In the presence of S9 mix, the mean mitotic index observed was 9.44 (NC), 9.70 (VC), 9.66 (T1), 9.42 (T2), 9.60 (T3) and 9.60 (PC).

In the cytotoxicity experiment, the highest test concentration0.125 (T1), 0.25 (T2) and 0.5 (T3)mg/ mLof culture mediashowed not more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence these concentrations were selected for the main study.

Hence, 0.5 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;

1.2                 

1.2.1             

In the experiment, the cultures were exposed to Licowax R 21 S FL 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 10.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.667 (T1), 0.667 (T2), 1.000 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/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.

During thetreatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 10.05, 9.72, 9.49, 9.53 and 9.56 andin the presence ofmetabolic activation were 9.70, 9.87, 9.60, 9.68 and 9.66 for NC, VC, T1, T2 and T3 concentrations respectively.

1.2.2             

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 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/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),0.333(T3) and 9.667 (PC) in the of absence metabolic activation and0.333(NC),0.333(VC), 1.000 (T1), 0.667 (T2), 0.667 (T3) and 10.667 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/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 mean mitotic indexin the absence of metabolic activation were 9.62, 9.66, 9.23, 9.47 and 9.33 andin the presence ofmetabolic activation were 9.59, 9.83, 9.51, 9.54 and 9.31 for NC, VC, 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.

Conclusion

In conclusion, under the experimental conditions and results of this study, it is concluded that Licowax R 21 S FL is“non-mutagenic”both in the presence (1% and 2%) and in the absence of metabolic activation in this assay

Conclusions:

under the experimental conditions and results of this study, it is concluded that Licowax R 21 S FL is “non-mutagenic” both in the presence (1% and 2%) and in the absence of metabolic activation in this assay

Executive summary:

                 

This study was conducted to determine the chromosomal aberration induction potential of Licowax R 21 S FL in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 26September 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, Ethyl alcohol 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 0.5 mg/mL concentration. Hence 0.5 mg/mL was selected as the highest concentration for cytotoxicity test.

Before conducting the chromosomal aberration study, Licowax R 21 S FL was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.125 (T1), 0.25 (T2) and 0.5 (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 10.49 (NC), 9.78 (VC), 9.44 (T1), 9.52 (T2), 9.58 (T3) and 9.21 (PC). In the presence of S9 mix, the mean mitotic index observed was 9.44 (NC), 9.70 (VC), 9.66 (T1), 9.42 (T2), 9.60 (T3) and 9.60 (PC).

In the cytotoxicity experiment, the highest test concentration0.125 (T1), 0.25 (T2) and 0.5 (T3)mg/ mLof culture mediashowed not more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence these concentrations were selected for the main study.

Hence, 0.5 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;

           

In the experiment, the cultures were exposed to Licowax R 21 S FL 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 10.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.667 (T1), 0.667 (T2), 1.000 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/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.

During thetreatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 10.05, 9.72, 9.49, 9.53 and 9.56 andin the presence ofmetabolic activation were 9.70, 9.87, 9.60, 9.68 and 9.66 for NC, VC, T1, T2 and T3 concentrations respectively.

     

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 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/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),0.333(T3) and 9.667 (PC) in the of absence metabolic activation and0.333(NC),0.333(VC), 1.000 (T1), 0.667 (T2), 0.667 (T3) and 10.667 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/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 mean mitotic indexin the absence of metabolic activation were 9.62, 9.66, 9.23, 9.47 and 9.33 andin the presence ofmetabolic activation were 9.59, 9.83, 9.51, 9.54 and 9.31 for NC, VC, 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.

Conclusion

In conclusion, under the experimental conditions and results of this study, it is concluded that Licowax R 21 S FL is“non-mutagenic”both in the presence (1% and 2%) and in the absence of metabolic activation in this assay

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jul 2016 - March 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:

other: in vitro mammalian cell transformation assay

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

Metabolic activation:

with and without

Metabolic activation system:

AroclorTM 1254 induced liver homogenate (S9)

Test concentrations with justification for top dose:

2, 1, 0.5,and 0.25 mg/mL both in the presence and absence of metabolic activation
based on the results of the pre test

Vehicle / solvent:

ethanol

Untreated negative controls:

yes

Remarks:

DMEM

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

TREATMENT
The culture flasks were incubated at 37°C in humidified atmosphere with 5% CO2 for 24 hours. After 24 hours, the medium was replaced with DMEM0 containing various concentrations of the test item either in the presence or absence of metabolic activation. Positive and solvent controls were performed in parallel. After 4 h the medium was replaced with complete medium following two washing steps with DMEM0 for Phase I and Phase II was performed with treatment duration of 24 hours in the absence of metabolic activation system and 4 hours in the presence of metabolic activation system.
The phenotypic expression was for a period of 7 days and then the cells were re-plated in medium with and without selective agent (6-thioguanine) for the determination of the number of mutants and cloning efficiency, respectively.
The colonies were fixed and stained with 10% methylene blue. The stained colonies with more than 50 cells were counted.

DATA RECORDING
The colonies were counted manually. The mean values of the colonies were calculated for each concentration together with standard deviation. Microsoft Office Excel based calculation was used for descriptive statistical analysis.

Rationale for test conditions:

conditions were chosen based on results of a pre test

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:

The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups using Mann-Whitney test. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Conclusions:

In conclusion it is stated that during the mutagenicity test described and under the experimental conditions reported the test items did not induce mutations in the HPRT locus in V79 cells of the Chinese hamster in the absence and presence of metabolic activation.
Therefore, Licowax R 21 S was considered to be “non-mutagenic” in this HPRT assay.

Executive summary:

This study was conducted to investigate the potential ofLicowax R 21 Sto 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 experiments, 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 with out metabolic activation.

2mg/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 both Phase - I and Phase - II based on cytotoxicity results.

2.00 mg/mL, 1.00 mg/mL , 0.50 mg/mL ,0.25 mg/mL both in the presence and absence of metabolicactivation.

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 and as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100%) with the RS for the test item in 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) was 5.27, 21.52, 11.06, 14.69, 19.10, 22.42, and 165.35/10⁶cells respectively in the absence of metabolic activation, number of mutant colonies of NC, VC, T1 ,T2 ,T3 ,T4 and PC (DMBA) was 6.08, 21.25, 14.84, 14.76, 21.72, 23.53 and 910.62 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) was 6.51, 24.62, 14.44, 14.69, 29.80, 27.06 and 175.27/10⁶cells respectively in the absence of metabolic activation, and the number of mutant colonies of NC, VC, T1 ,T2 ,T3 ,T4 and PC (DMBA) was 14.38, 23.07, 20.72, 25.47, 21.74, 22.75, and 1100.88 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) was 6.0, 18.6, 15.7, 24.5, 30.7, 41.2, and 261.0/10⁶cells respectively in the absence of metabolic activation, and the number of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) was 6.2, 18.4, 18.4, 27.3, 36.0, 40.2, and 1450.6./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 4.7, 15.4, 16.2, 19.4, 23.1, 30.3, and 199.0/10⁶cells respectively in the absence of metabolic activation, and the number of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) was 6.3, 13.9, 14.0, 20.5, 23.8, 32.8, and 1067.8 per 10⁶cells respectively in presence of metabolic activation.

In both the cultures, there was no distinct increase in the mutant frequency ofLicowax R 21 Swhen 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 were 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 experimentLicowax R 21 Sdoes not show a distinct increase in the number of mutant colonies and thus proved the validity of test system and activity of the S9 mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In conclusion, Licowax R21 S is not mutagenic in the bacterial reverse mutation assay, thein vitrogene mutation study in Chinese hamster V79 cells and thein vitrochromosome aberration in human lymphocytes in the presence and absence of metabolic activation up to the tested concentrations.

Justification for classification or non-classification

The registration substancedoes not have to be not 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 the in vitro gene mutation assay or in the in vitro chromosome aberration study in human lymphocytes.