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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

73138-58-6,73138-59-7,73138-60-0

IUPAC Name:

73138-58-6,73138-59-7,73138-60-0

Test material form:

solid: flakes

Details on test material:

- Name of test material (as cited in study report): Licowax R 21 S FL
- CAS Number : 73138-58-6,73138-59-7,73138-60-0
- Molecular weight (if other than submission substance): Guerbet reaction Products: 505.6,
Fatty acids, tallow, Guerbet reaction Products, Ca salts: 1049.33, Fatty acids, tallow, Guerbet reaction Products, Na salts: 527.6
- Structural formula attached as image file (if other than submission substance): see attach-1
- Substance type: Fatty acids, tallow, Guerbet reaction Products,
- Physical state:Slightly yellow flakes
- Analytical purity: 99.6 % (w/w)
- Lot/batch No.: DEF2084336
- Expiration date of the lot/batch: November 13, 2018
- Stability under test conditions: Unknown
- Storage condition of test material: Room Temperature (20 to 30oC)

Method

Target gene:

Histidine

Species / strain

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

Controls

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

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.