Fatty acids, tall-oil, polymers with 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

Study period:

From July 14, 2015 to July 30, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl (9Z)-octadec-9-enoate; 2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl (9Z,12Z)-octadeca-9,12-dienoate; 2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl octadecanoate

EC Number:

617-001-2

Cas Number:

80207-00-7

Molecular formula:

not applicable

IUPAC Name:

2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl (9Z)-octadec-9-enoate; 2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl (9Z,12Z)-octadeca-9,12-dienoate; 2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl octadecanoate

Test material form:

liquid

Remarks:

Brown

Method

Target gene:

hisD6610- frame shift (histidine deficiency)
hisD3052- frame shift (histidine deficiency)
hisG46- base pair substitution (histidine deficiency)
hisG428- base pair substitution (histidine deficiency)
uvrB- deletion UV sensitivity (biotine deficiency)
rfa- deletion (lipopolysaccharide side chain deficiency)
pKM101- plasmide (ampicillin resistance)
pAQ1- plasmide (tetracycline resistance)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: S. typhimurium TA 97a

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 with Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535) (plate incorporation method)
0, 50, 150, 500, 1500, 5000 μg/plate

Experiment 2a: bacteria strains TA102 and TA1535 (pre-incubation method)
0, 78, 156, 313, 625, 1250, 2500, 5000 μg/plate

Experiment 2b: bacteria strains TA97a, TA98 and TA100 (pre-incubation method)
0, 78, 156, 313, 625, 1250, 2500, 5000 μg/plate

Vehicle / solvent:

Ethanol

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Amino-Anthracene

Remarks:

with metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine

Remarks:

without metabolic activation

Details on test system and experimental conditions:

CULTURE OF BACTERIA
8 h before the start of each experiment, one lyophilisate per strain to be used was taken from the fridge to inoculate a culture vessel containing nutrient broth. For the incubation of strains TA97a, TA98, TA100, TA102 ampicilline was added to the nutrient broth (25 mg/L), for the incubation of strain TA102, tetracycline was added (2 mg/L) in addition to ampicilline. TA1535 was incubated without the addition of antibiotics. The flasks were incubated at 37±1°C for 8 h.

CONDUCT OF EXPERIMENT
Description of method: Per strain and dose, 3 plates with and 3 plates without S9 mix were used. The test substance solutions were prepared from stock solution containing 50 g/L in ethanol. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine biotin solution 0.5 mM per 100 mL basis was added and the bottle was placed in the water bath at 43±1°C.

Plate incorporation method:
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
• 100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
• 500 μL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 μL bacteria suspension
• 2,000 μL overlay agar (top agar)
The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37±1°C.

Pre-incubation method:
The following materials were gently vortexed in a test tube and incubated at 37±1°C for 20 min:
• 100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
• 500 μL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 μL bacteria suspension
After pre-incubation, 2,000 μL overlay agar (top agar) was added, the tube was gently vortexed and the mixture was poured onto the selective agar plate. The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37±1°C.

Evaluation criteria:

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

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: T97a, TA 98, TA 100, TA 102, 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

Additional information on results:

Experiment 1:
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test substance was stated as not mutagenic under the test conditions.

To verify this result, a second experiment was performed using the pre-incubation method.
Experiment 2a and b:
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed with one exception.
In the highest concentration (5,000 μg/plate) the number of revertant colonies of the strain TA1535 showed an increase. But this can be seen as uncritical, because the difference is marginal and no concentration-related increase over the tested range was found.
Therefore, the test substance was stated as not mutagenic under the test conditions.

Acceptability of study:
Nearly all spontaneous revertants and all positive control values were within the range of the historical data. Difference of revertants lying outside the range are marginal. Therefore, the study was considered valid.

Applicant's summary and conclusion

Conclusions:

No mutagenic effect of test substance was observed either in the presence or absence of metabolic activation system under the conditions of this bacterial reverse mutation assay.

Executive summary:

An in vitro bacterial reverse mutation assay was performed to test the potential of the substance to cause gene mutations according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. The study included 3 valid experiments. In experiment 1, the plate incorporation method was used for 5 concentrations (0, 50, 150, 500, 1500, 5000 μg/plate) and in the experiments 2a and b, the pre-incubation method was used for 7 concentrations (0, 78, 156, 313, 625, 1250, 2500, 5000 μg/plate). The experiments were carried out using strains of Salmonella typhimurium (TA 97a, TA98, TA100, TA 102 and TA 1535) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of Aroclor 1254 induced rats. The colony numbers on the negative (vehicle) / positive control and test substance treated plates were counted visually and the numbers were recorded. In all 3 experiments, no significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration related increase over the tested range was observed with one exception. In experiment 2, the highest concentration (5000 μg/plate) showed an increase in number of revertant colonies of the strain TA1535. This was considered not critical as the difference was marginal and no concentration-related increase over the tested range was found. No mutagenic effect of the test substance was observed either in the presence or absence of metabolic activation system under the conditions of this bacterial reverse mutation assay (Andres, 2015).