Fatty acids, linseed-oil, reaction products with bisphenol A-epichlorohydrin polymer and glycidyl neodecanoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of oxiran-2-ylmethyl 2,2,3,5-tetramethylhexanoate with linseed oil fatty acids (including majorly linolenic acid, linoleic acid and oleic acid) (1:1)

Molecular formula:

Not applicable (UVCB constituent)

IUPAC Name:

Reaction product of oxiran-2-ylmethyl 2,2,3,5-tetramethylhexanoate with linseed oil fatty acids (including majorly linolenic acid, linoleic acid and oleic acid) (1:1)

Constituent 2

Reference substance name:

Reaction product of 4,4'-Methylendiphenyldiglycidylether (BADGE) with linseed oil fatty acids (including majorly linolenic acid, linoleic acid and oleic acid) (1:2)

Molecular formula:

Not applicable (UVCB constituent)

IUPAC Name:

Reaction product of 4,4'-Methylendiphenyldiglycidylether (BADGE) with linseed oil fatty acids (including majorly linolenic acid, linoleic acid and oleic acid) (1:2)

Constituent 3

Reference substance name:

Reaction product of 4,4'-Methylendiphenyldiglycidylether (BADGE) with linseed oil fatty acids (including majorly linolenic acid, linoleic acid and oleic acid) (1:1)

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Reaction product of 4,4'-Methylendiphenyldiglycidylether (BADGE) with linseed oil fatty acids (including majorly linolenic acid, linoleic acid and oleic acid) (1:1)

Constituent 4

Reference substance name:

Reaction product of 4,4'-Methylendiphenyldiglycidylether (BADGE) with linseed oil fatty acids (including majorly linolenic acid, linoleic acid and oleic acid) (1:3)

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Reaction product of 4,4'-Methylendiphenyldiglycidylether (BADGE) with linseed oil fatty acids (including majorly linolenic acid, linoleic acid and oleic acid) (1:3)

Constituent 5

Reference substance name:

High Mw components based on linseed oil fatty acids

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

High Mw components based on linseed oil fatty acids

Constituent 6

Reference substance name:

Sum of unassigned components

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Sum of unassigned components

Test material form:

liquid

Details on test material:

UVCB

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch number 210162718
- Expiration date of the lot/batch: 16-12-2017
- Purity: 100% (UVCB)
- Purity test date: Not reported


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Stability in dimethyl sulfoxide (DMSO) - 96 h, solubility in DMSO >1 g/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Dilution of the test material in the vehicle: The test substance was dissolved in DMSO to produce a stock solution of 50 g/L and from this stock, serial dilutions of the working solutions were prepared in DMSO to result in nominal concentrations of 5000, 1500, 500, 150 and 50 μg/plate.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 97

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

5000, 1500, 500, 150 and 50 μg/plate for plate incorporation experiment
5000, 2500, 1250, 625 and 313 μg/plate for pre-incubation experiment

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO does not have any effect on the viability of the bacterial strains or the number of spontaneous revertants in the tested concentrations and the test substance is sufficiently soluble in the solvent at >1 g/L

Controls

Untreated negative controls:

yes

Remarks:

The solvent DMSO was used as the negative control

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-nitro-1,2-phenylene diamine, 2-amino anthracene

Remarks:

Sodium azide and 4-nitro-1,2-phenylene diamine were used without metabolic activation. Benzo(a)pyrene and 2-aminoanthracene were used in presence of metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Two experiments were conducted. A plate incorporation method was used for the first experiment, followed by the second experiment (pre-incubation method).

FIRST EXPERIMENT (plate incorporation menthod)

DURATION
- Preincubation period: Not reported
- Exposure duration: 48 h

NUMBER OF REPLICATES USED: Per strain and dose, 3 plates with and 3 plates without S9 mix were used.

METHOD OF APPLICATION

The following solutions/suspensions were gently vortexed in a test tube and poured onto the selective agar plates:
1. 100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
2. 500 μL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation)
3. 100 μL suspension of the bacterial strains
4. 2000 μL overlay agar on the top
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37±1°C.



SECOND EXPERIMENT (pre-incubation menthod)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATES USED: Per strain and dose, 3 plates with and 3 plates without S9 mix were used.

METHOD OF APPLICATION

The following materials were gently vortexed in a test tube and incubated at 37±1°C for 20 min:
1. 100 μL test substance suspension at each dose level, solvent (negative control) or reference
mutagen solution (positive control)
2. 500 μL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
3. 100 μL suspension of the bacterial strains
After pre-incubation, 2000 μL overlay agar was added to the top, the tube was gently vortexed and the mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37±1 °C.

TOXICITY CONTROL
Performed in experiment 1 only, analogous to the titre control with the maximum dose of test substance with and without S9 on maximal-soft agar, 2 replicates with and without metabolic activation, incubation for 48 h at 37±1°C.

POSITIVE CONTROL
For all the positive control substances, 3 replicates were prepared. The stock solutions of the positive control substances were diluted to effect an application volume of 0.1 mL/plate, incubation for 48 h at 37 ±1°C.

Evaluation criteria:

A test substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

Microsoft Excel® spreadsheet was used to calculate mean values and standard deviations of each treatment concentration, solvent control and positive control.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 97, 98, 100, 102, 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: in both pre-incubation and plate incorporation experiments

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test substance was considered to be non-mutagenic to the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation.

Executive summary:

An in vitro bacterial reverse mutation study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. Two experiments were performed.In the first experiment, five concentrations of the test substance, dissolved in DMSO (ranging from 50 to 5000 µg/plate) were used. Five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535) were exposed to the test substance both in the presence and in the absence of a Aroclor induced rat liver S9-mix metabolic activation system for 48 h, using theplate incorporation method.None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test substance showed no precipitates on the plates in all tested concentrations. To verify the results of the first experiment, a second experiment was performed, using 5 concentrations of the test substance (ranging from313to 5000 µg/plate) and a modification in study performance (i.e., using the pre-incubation method). The test substance did not show mutagenic effects in the second experiment, either. The test substance also showed no precipitates on the plates in all tested concentrations. Further, in both the experiments, no signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were also in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. The study was therefore considered valid.Therefore, the test substance was considered to be non-mutagenic under the conditions of the reverse mutation assay (Andres, 2016).