Esterification product of sunflower-oil fatty acids, with 1,4:3,6-dianhydro-D-glucitol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 December 2015 to 1 February 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No preincubation test was performed. No certificate of analysis was provided.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

1818326-42-9

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot #CB 15026
- Expiration date of the lot/batch: Not detailed
- Purity test date: Not detailed

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not detailed
- Stability under test conditions: Not detailed
- Solubility and stability of the test substance in the solvent/vehicle: Not detailed
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not detailed

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was diluted in dimethyl sulfoxyde and tested.
- Preliminary purification step (if any): Not applicable
- Final dilution of a dissolved solid, stock liquid or gel: Not detailed
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Test substance was diluted in DMSO

Method

Species / strain

Species / strain / cell type:

other: S. Typhimurium TA97a, TA98, TA100, TA102 and TA1535

Additional strain / cell type characteristics:

other: uvrB mutation, absence of R factor plasmid, rfa mutation

Metabolic activation:

with and without

Metabolic activation system:

The S9 activation system was used to screen for the presence of mutagens from byproducts of the test article. Rat liver S-9 homogenate was obtained from Molecular Toxicology, Inc.

Test concentrations with justification for top dose:

Concentrations for test item used were : 5, 1.6, 0.5, 0.16 and 0.05 µL/plate. The concentrations were based on the highest recommended concentration for non cytotoxic compounds (based on OECD 471)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:no justification was provided

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

mitomycin C

other: 4-nitro-0-phenylene-diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) ; Spot test
- Cell density at seeding (if applicable):100 µL from stock 10E8 CFU/mL

DURATION
- Preincubation period: not applicable
- Exposure duration: 48-72 hours
- Expression time (cells in growth medium): during exposure : 48-72 hours
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48-72 hours

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): not applicable

STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: triplicates were used per condition

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable

NUMBER OF CELLS EVALUATED: at least 300 Colony forming unit were counted

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Not applicable

DETERMINATION OF CYTOTOXICITY
- Method: Not detailed

Spot tests; The test article was also analyzed using the spot method on plates with and without metabolic activation system. Two mL aliquots of the top agar and 100 µL of the appropriate test organism were added tominimal glucose agar plates. The plates were allowed to harden then 10µL of the test article was added as a spot on the surface of the plate. The plates were incubated for growth of the organisms at 37°C ± 2°C for 48-72 hours. Only the highest test article concentration was analyzed using the spot method

Evaluation criteria:

Criteria for mutagen determination:
- A reversion rate greater than 200% of the solvent control in strains TA97a, TA100 and TA102. A reversion rate greater than 300% of the solvent control in strain TA1535 and TA98
- Demonstration of a clear dose related response when dilutions are tested.

Criteria for non mutagen determination:
- A reversion rate lass than or equal to 20% of the solvent control in strains TA97a, TA100 and TA102. A reversion rate less than or equal to 300% of the solvent control in strains TA98 and 1535
- No dose related response when dilutions are tested

Results and discussion

Test results

Key result

Species / strain:

other: S. Typhimurium TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test article concentrations did not produce a two fold or three fold increase of the number of revertants or produce a clear dose related response in any of the 5 tester strains. The spot tests showed no zone of increased reversion or of toxicity

Applicant's summary and conclusion

Conclusions:

Under the experimental condition of the study, the test item HydraSynol IDL did not meet criteria for a potential mutagen for bacteria. Hence the test item was not considered as mutagenic for bacteria strain according to CLP criteria.

Executive summary:

This GLP compliant study was performed to determine the potential mutagenic activity of the test article HydraSynol IDL with a method equivalent to OECD TG 471 (Ames test).

The Ames test employs several histidine dependent (His-) strains for S. Typhimurium which require the amino acid histidine for growth. The test detects mutations which cause the bacterial strains to revert to histidine independant (His+) bacteria which are capable to growth in absence of histidine. The assays used tester strains TA97a, TA98, TA100, TA102 and TA1535 which were selected to detect various types of mutagens. The S-9 activation system was designated to simulated mammalian liver enzyme systems and is used to detect substances which undergo metabolic activation from non mutagenic forms.

Plate incorporation method was used. The test item was diluted in DMSO and was applied at different concentrations : 5, 1.6, 0.5, 0.16 and 0.05 µL/plate. Bacteria were incubated for 48 - 72 hours with test item and were counted for revertant colony. Spot test was performed to evaluate potential cytotoxicity.

The test article concentrations did not produce a two fold or three fold increase of the number of revertants or produce a clear dose related response in any of the 5 tester strains. The spot tests showed no zone of increased reversion or of toxicity

Under the experimental condition of the study, the test item HydraSynol IDL did not meet criteria for a potential mutagen for bacteria. Hence the test item was not considered as mutagenic for bacteria strain according to CLP criteria.