Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-08-01 to 2017-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

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:

Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

EC Number:

279-510-2

EC Name:

Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

Cas Number:

80584-99-2

Molecular formula:

n.a.

IUPAC Name:

Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

Test material form:

liquid

Details on test material:

- Batch No: 3300081
- Physical state: liquid
- Colour: colourless to yellow
- Purity: 100 %
- Expiry date: 2018-05-16
- Storage conditions: Room temperature (20 ± 5 °C), keep away from light

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1525

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem GmbH (batch of the bacteria strains: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) and were stored as lyophilisates in the fridge at 2-8 °C.
The lyophilisates were used to prepare permanent cultures which were filled into vials and stored at < - 75 °C.

MEDIA USED:
- Nutrient Broth for Overnight Culture
Nutrient broth Merck 5443: 2.8 g
H2O demineralised: ad 350 mL
- Isotonic Sodium Chloride Solution for Dilution Purposes
Sodium chloride: 0.9 g
H2O demineralised: ad 100 mL
- Vogel-Bonner-Medium 20fold
Magnesium sulphate (MgSO4*7H2O): 4.0 g
Citric acid mono hydrate (MR 210.14 g/mol): 40.0 g
Potassium phosphate, dibasic (anhydrous) (K2HPO4): 200.0 g
Sodium ammonium phosphate, monobasic, tetra hydrate (Na(NH4)HPO4*4H2O): 70.0 g
H2O demineralised: ad 1000.0 mL
- Glucose Solution 40%
Glucose monohydrate (MR 198.17g/mol): 440.0 g
H2O demineralised: ad 1000.0 mL
- Minimal Glucose Agar
Vogel-Bonner-Solution 20fold: 500.0 mL
Glucose solution 40%: 500.0 mL
H2O demineralised: 9000.0 mL
Agar: 150.0 g
- Biotin Agar
Minimal-Glucose-Agar. 80 °C: 500.0 mL
Biotin solution 0.5 mM: 3.0 mL
- Histidine-Biotin-Agar
Biotin-Agar, 80 °C: 350.0 mL
Histidine solution 0.5%: 3.5 mL

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Metabolic activation system

Test concentrations with justification for top dose:

- Experiment I: 5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate and 50 μg/plate.
- Experiment II: 5000 μg/plate, 2500 μg/plate, 1250 μg/plate, 625 μg/plate, 313 μg/plate and 156 μg/plate.

Vehicle / solvent:

- Vehicle used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: Based on the results from a solubility test: In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralised H2O, dimethyl sulfoxide (DMSO), ethanol and in a concentration of 200 g/L in tetrahydrofuran (THF). THF was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535 (1 µg/plate, without S9)

Controls 2

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA98 (20 µg/plate), with metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Amino-Anthracene

Remarks:

TA97a, TA100, TA102 and TA1535 (1 µg/plate), with S9

Controls 4

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Remarks:

TA97a, TA98 and TA102 (20 µg/plate), without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, Experiment I); preincubation (Experiment II)

EXPERIMENTAL PERFORMANCE
Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nu-trient broth. After incubation for eight hours at 37 ± 1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre. Per strain and dose, three plates with and three plates without S9 mix were used. 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.

- Experiment I :
Date of treatment: 02. Aug. 2016
Concentrations tested: 5000 / 1500 / 500 / 150 / 50 µg/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tester strains: TA97a, TA98, TA100, TA102, TA1535
Method: plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
- 25 µL test solution at each dose level, 100 µL solvent (negative control) or refer-ence mutagen solution (positive control)
- 500 µL S9 mix (see chapter 6.4.18, for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
- 100 µL bacteria suspension (see chapter 6.3.2, test system, culture of the strains)
- 2000 µL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

- Experiment II:
Date of treatment: 09. Aug. 2016
Concentrations tested: 5000 / 2500 / 1250 / 625 / 313 / 156 µg/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tester strains: TA97a, TA98, TA100, TA102, TA1535
Method: pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
- 25 µL test solution at each dose level, 100 µL solvent (negative control) or refer-ence mutagen solution (positive control)
- 500 µL S9 mix (see chapter 6.4.18, for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
- 100 µL bacteria suspension (see chapter 6.3.2, test system, culture of the strains)
After pre-incubation, 2000 µL overlay agar (top agar) was added, the tube was gently vor-texed 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.

EVALUATION:
The colonies were counted visually and the numbers were recorded. A spreadsheet soft-ware (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given. A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant 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.

Evaluation criteria:

- The colonies were counted visually and the numbers were recorded.
- A 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:

- The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the first experiment, VOELOFA Monomer (dissolved in THF) was tested up to concen-trations of 5000 µg/plate in the absence and presence of S9-mix (0.74 % final concentration in the treatment) in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. VOELOFA Monomer showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no rele-vant decrease in the number of revertants was observed in all bacteria strains. The test item VOELOFA Monomer showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.
On the base of the first experiment, VOELOFA Monomer was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix (0.74% final concentration in the treatment) in all bacteria strains using the pre-incubation method. VOELOFA Monomer showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item VOELOFA Monomer showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiments showed that the test item VOELOFA Monomer caused no increase in the number of revertants in all bacteria strains compared to the solvent con-trol, in both the absence and presence of metabolic activation. The test item VOELOFA Monomer did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Remarks on result:

other: Experiment I

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, VOELOFA Monomer did not cause gene mutations in an Ames Test conducted according to OECD 471. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse gene mutation assay.

Executive summary:

In a bacterial reverse gene mutation assay conducted according to OECD guideline 471, strains TA97a, TA98, TA100, TA102 and TA1535 of Salmonella typhimurium were exposed to VOELOFA Monomer (100% purity) in THF at concentrations of up to 5000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains in both experiments (plate incorporation and pre-incubation). Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.