C16-18 (even numbered) and C18 (unsaturated) triglycerides, hydroxylated, oxidatively cleaved, hydrolised (glycerol free).

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 April 14th to April 29th, 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 26. Jun. 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 30. May 2008

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:

C16-18 (even numbered) and C18 (unsaturated) triglycerides, hydroxylated, oxidatively cleaved, hydrolised (glycerol free).

EC Number:

701-347-7

Molecular formula:

From C9H1802 to C24H48O2

IUPAC Name:

C16-18 (even numbered) and C18 (unsaturated) triglycerides, hydroxylated, oxidatively cleaved, hydrolised (glycerol free).

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: histidine deficiency, UV sensitivity, biotin deficiency, lipopolysac-charide side chain deficiency, ampicillin resistance, tetracycline resistance

Remarks:

All Salmonella typhimurium strains were obtained from Trinova BioChem GmbH (batch: TA98: 5491D and 5508D, TA100: 5506D, TA102: 5457D, TA1535: 5504D, TA1537: 5503D and 5530D) and were stored as lyophilizates in the refrigerator at 2 – 8 °C.

Metabolic activation:

with and without

Metabolic activation system:

S9 was obtained by Trinova Biochem GmbH, Gießen.
Batch nos. 4344
Specification produced from the livers of male Sprague-Dawley rats which were treated with phenobarbital/5,6-Benzoflavone.

S9-mix
Phosphate buffer 22.5 mL
0.1M NADP-solution 1.0 mL
1M G6P-solution 0.125 mL
Salt solution 0.5 mL
Rat liver S9 1.0 mL

Test concentrations with justification for top dose:

The following nominal test item concentrations were prepared for experiment 1 (plate incorporation):
• TA98, TA100, TA102, TA1535, TA1537: 5000, 1500, 500, 150 and 50 µg/plate.

The following nominal test item concentrations were prepared for experiment 1b (plate incorporation):
• TA1535: 5000, 1500, 500, 150, 50, 15, 5 µg/plate
• TA98, TA100, TA1537 (+S9): 1500, 500, 150, 50, 15, 5, 1.5 µg/plate
• TA1537 (-S9): 150, 50, 15, 5, 1.5, 0.5, 0.15 µg/plate

The following nominal test item concentrations were prepared for experiment 1c (plate incorporation):
• TA1535: 5000, 1500, 500, 150, 50, 15, 5 µg/plate

The following nominal test item concentrations were prepared for experiment 2 (pre-incubation method):
• TA102, TA1535: 5000, 2500, 1250, 625, 312.5, 156.3 and 78 µg/plate
• TA98, TA100, TA1537 (+S9): 1500, 750, 375, 188, 93.8, 46.9, 23 µg/plate
• TA1537 (-S9): 150, 75, 37.5, 18.8, 9.4, 4.7, 2.3 µg/plate

Experiment 1b and 1c were conducted using lower concentration of the test item with respect to experiment 1 because of the observed toxicity of the test material at that concentrations.

Vehicle / solvent:

DMSO (Dimethylsulfoxide) was used for the test item and for for the positive controls 4-Nitro-1,2-phenylene diamine, benzo-a-pyrene and 2-amino-anthracene.
Demineralized water, prepared in the laboratory, from an ion-exchanger, batch: T20210120 was used as solvent for the positive control sodium azide.
Sodium chloride (0.9 % NaCl), prepared in the laboratory for the positive control Mitomycin C (MMC), batch: T20210408 (exp. 1, 1c, 2), T20210325 (exp. 1b) for the positive control MMC

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

mitomycin C

other: 4-Nitro-1,2-phenylene diamine in DMSO, without metabolic activation and 2-Amino-anthracene in DMSO, with metabolic activation

Details on test system and experimental conditions:

- Number of cultures per concentration: Per bacteria strain and concentration, three plates with (+S9) and three plates without met-abolic activation (-S9) were used
- Number of independent experiments: 4 in particular, experiments 1, 1b, 1c used plate incorporation method whereas experiment 2 used pre incubation method
- Cell density: around 10^9 cells/mL

Preaparations
On the day before the start of each experiment, a nutrient broth (Oxoid nutrient broth no. 2) was inoculated with one lyophilizate per strain at 3:30 pm. These overnight cultures were placed in the heating chamber at 37 ± 1 °C for 16.5 hours. For the last two hours the overnight cultures were shaken on an orbital shaker (150 rpm) at 37 ± 1 °C. Afterwards, the overnight cultures were ready for use in the experiment. During the test, the overnight cultures were stored at room temperature (20 ± 5 °C) to prevent changes in the titre.
Per bacteria strain and concentration, three plates with (+S9) and three plates without metabolic activation (-S9) were used.
Different media and solutions were prepared preliminary (exact production dates are docu-mented in the raw data).
On the day of the test, the bacteria cultures were checked for growth visually. The incubation chambers were heated to 37 ± 1 °C. The water bath was turned to 43 ± 1 °C. The table surface was disinfected.
The S9-mix was freshly prepared and stored at 0 °C

- Incubation time: 48 h
- Incubation temperature: 37 ± 1 °C
- For the top agar, 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture 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). For the positive control MMC, 2.5 μL of the stock solution were applied to achieve a final concentration of 0.5 μL/plate.
- 500 μL S9-mix (see chapter 6.4.13, page 18 for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
- 100 μL bacteria suspension
- 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.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ± 1 °C for 20 minutes:
- 100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control). For the positive control MMC, 2.5 μL of the stock solution were applied to achieve a final concentration of 0.5 μL/plate.
-500 μL S9-mix (see chapter 6.4.13, page 18 for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
- 100 μL bacteria suspension
After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently slewed. 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 incubator at 37 ± 1 °C.

References and Validity
- Genotype Confirmation: Confirmation of genotype is performed for each batch of lyophilized bacteria by the supplier. The batches used of lyophilized bacteria met the criteria
- Spontaneous Revertants: The number of spontaneous revertants was determined for each solvent used in the test by investigating three replicates with and without metabolic activation, incubation for 48 hours at 37 ± 1 °C for each strain.
- Determination of Titre: The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on nutrient agar. It should give a density of 10^9 cells/mL (at the least). Two replicates with and without metabolic activation, incubation for 48 hours at 37 ± 1 °C.
- Toxicity Control: Performed in experiment 1 only and analogously to the titre control with the maximum dose of the test item on nutrient agar. Two replicates with and without metabolic activation, incubation for 48 hours at 37 ± 1 °C.
- Sterility Control: Performed simultaneously and exactly as the test item but with solvent only and S9 (without adding bacteria) on top agar. Four replicates, incubation for 48 hours at 37 ± 1 °C.
- Solubility: Plates were checked for precipitation of test item at the end of the incubation by visual inspection.
- Positive Controls: Using diagnostic mutagens. The stock solutions of the substances were diluted to achieve an application volume of 0.1 mL/plate.
Three replicates with and without metabolic activation, incubation for 48 hours at 37 ± 1 °C.

- Evaluation: Five different analysable and non-toxic concentrations were used for the evaluation of the mutagenic potential of the test item.
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, negative control and positive control.
The mean values and standard deviations of each threefold determination were calculated as well as the increase factor 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 (mean revertants minus mean spontaneous revertants) is given.

Evaluation criteria:

A result is considered as positive if a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the concentrations occurs in at least one tested strain with or without metabolic activation.

A biologically relevant increase is described as follows :
• if in the bacteria strains S. typhimurium TA98, TA100, TA102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase fac-tor of at least 2.0)
• if in the bacteria strains S. typhimurium TA1535 and TA1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3.0).

A substance is not mutagenic if it does not meet the criteria above.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

citotoxicity of the test item was observed for TA100 (+/-S9) at 5000 and 1500 µg/plate in experiment 1. Experiment 1b was conducted in the same conditions with lower concentration of the test item.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Citotoxicity was observed TA98 (+/-S9) at 5000 and 1500 µg/plate in experiment 1. Experiement 1b was conducted in the same condition with lower concentrations of the test item.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Citotoxicity was observed for TA1537 (-S9) at 5000, 1500, 500 and 150 µg/plate and for TA1537 (+S9) at 5000 and 1500 µg/plate. The experiment was repeted (1b) with lower concentrations in the same conditions.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

citotoxicity was observed for TA 1535 (-S9) at test item concentration of 5000 µg/plate in experiment 1. Therefore the experiment was repeted under the same test conditions with lower concentrations in experiemnt 1b.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 1
- Confirmation of the Criteria and Validity
All strains met the criterion of at least 10^9 bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (mean ± 3 standard deviations). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.
- Solubility and Toxicity: In the first experiment, the test item showed no precipitates on the plates in all tested concentrations. The bacterial background lawn was visible and not affected. The number of spontaneous revertant colonies was relevantly reduced for TA98 (+/-S9) at 5000 and 1500 μg/plate, for TA100 (+/-S9) at 5000 and 1500 μg/plate, for TA1535 (-S9) at 5000 μg/plate, for TA1537 (-S9) at 5000, 1500, 500 and 150 μg/plate and for TA1537 (+S9) at 5000 and 1500 μg/plate. Thus, the test item shows signs of toxicity towards these strains in the given concentrations. Towards TA102, and in the lower concentrations no signs of toxicity could be observed.
- Mutagenicity: No relevant or concentration-related increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. Experiment 1 had to be repeated for TA98, TA100, TA1535 and TA1537 due to toxic effects and therefore an insufficient number of analyzable non-toxic concentrations as indicated in the guideline.
Based on the toxicity results, this experiment was repeated under the same conditions with lower concentrations

Experiment 1b
- Confirmation of the Criteria and Validity: All strains (except from TA1535) met the criterion of at least 10^9 bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (mean ± 3 standard deviations). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic acti-vation and all were within the historical control data ranges. For TA1535 the experiment was not evaluated, as it was declared as invalid because of probable contamination.
- Solubility and Toxicity: In exp. 1b, the test item showed no precipitates on the plates in all tested concentrations. The bacterial background lawn was visible and not affected. The number of spontaneous revertant colonies was relevant reduced for TA100 (+/-S9) at 1500 μg/plate and for TA1537 (+S9) at 1500 μg/plate. Thus, the test item shows signs of toxicity towards these strains in the given concentrations. In the lower concentrations no signs of toxicity could be observed.
- Mutagenicity: No relevant or concentration-related increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. For TA1535 the experiment was not evaluated, as it was declared as invalid because of probable contamination.
Therefore, the test item is stated as not mutagenic under the conditions of this experiment in all evaluated strains. The experiment was repeated for TA1535 in exp. 1c

Experiment 1c
- Confirmation of the Criteria and Validity: The strain TA1535 met the criterion of at least 10^9 bacteria/mL (correlating to 100 colo-nies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (mean ± 3 standard deviations). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.
- Solubility and Toxicity: In exp. 1c, the test item showed no precipitates on the plates in all tested concentrations. The bacterial background lawn was visible and not affected. The number of spontaneous revertant colonies was not reduced. Thus, the test item showed no signs of toxicity towards these strains in the given concentrations.
- Mutagenicity: No relevant or concentration-related increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. Therefore, the test item is stated as not mutagenic in TA1535 under the conditions of this experiment.

Experiment 2
- Confirmation of the Criteria and Validity: All strains met the criterion of at least 10^9 bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (mean ± 3 standard deviations). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.
- Solubility and Toxicity: In exp. 2, the test item showed no precipitates on the plates in all tested concentrations. The bacterial background lawn was visible and not affected. The number of revertant colonies was relevant reduced for TA1535 (+/-S9) at 5000 μg/plate for TA1537 (+S9) at 1500 and 750 μg/plate and for TA1537 (-S9) at 150 μg/plate. Thus, the test item showed signs of toxicity towards these strains in the given concentrations. In the lower concentrations no signs of toxicity could be observed.
- Mutagenicity: No relevant or concentration-related increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. Therefore, the test item is stated as not mutagenic under the conditions of this experiment.

Validity
All negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
All positive controls showed f(I) values > 2 or > 3 (strain specific threshold) which demonstrated the mutagenic potential of the diagnostic mutagens.
The confirmation tests of the genotype performed did not show any irregularities. The control of the titre was above the demanded value of 10^9 bacteria/mL. In the sterility control no growth of bacteria could be detected.
Since all criteria for acceptability have been met, the study is considered valid.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that the substance is not mutagen-ic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

Executive summary:

The test item was tested in the Bacterial reverse mutation assay (based on OECD guideline 471) with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537).

The study was performed with the plate incorporation (exp. 1, 1b and 1c) and pre-incubation method (exp. 2) in the absence and presence of a metabolic activation system (S9). Under these conditions the influence of the test item on bacterial test strains was evaluated for mutagenicity.

The test item was dissolved in DMSO and showed no precipitates on the plates at any of the concentrations in all four experiments.

The exp. 1 had to be repeated for TA98, TA100, TA1535 and TA1537 due to toxic effects and therefore an insufficient number of analyzable non-toxic concentrations as indicated in the guideline. Experiment 1b was invalid for TA1535 because of probable contamination and was therefore repeated in exp. 1c.

In exp. 1, the number of spontaneous revertant colonies was relevant reduced for TA98 (+/-S9) at 5000 and 1500 µg/plate, for TA100 (+/-S9) at 5000 and 1500 µg/plate, for TA1535 (-S9) at 5000 µg/plate, for TA1537 (-S9) at 5000, 1500, 500 and 150 µg/plate and for TA1537 (+S9) at 5000 and 1500 µg/plate. Thus, the test item shows signs of toxicity towards these strains in the given concentrations. Towards TA102, and in the lower concentrations no signs of toxicity could be observed. Therefore experiment 1b was conducted in the same conditions with lower concentrations of the test item.

Clear signs of toxicity were observed for TA100 and TA1537 in exp. 1b: the number of spontaneous revertant colonies was relevant reduced for TA100 (+/-S9) at 1500 µg/plate and for TA1537 (+S9) at 1500 µg/plate. Thus, the test item shows signs of toxicity towards these strains in the given concentrations. In the lower concentrations no signs of toxicity could be observed.

In exp. 1c, no signs of toxicity could be observed towards TA1535.

Clear signs of toxicity were observed for TA1535 and TA1537 in exp. 2: in particular, the number of revertant colonies was relevant reduced for TA1535 (+/-S9) at 5000 µg/plate for TA1537 (+S9) at 1500 and 750 µg/plate and for TA1537 (-S9) at 150 µg/plate. Thus, the test item shows signs of toxicity towards these strains in the given concentrations. In the lower concentrations no signs of toxicity could be observed.

At least five non-toxic concentrations were evaluated for mutagenicity with the plate incorporation and the pre-incubation method with and without metabolic activation for all tested bacteria strains.

The results of all experiments showed that the test item caused no relevant or dose-related increase in the number of revertants in all bacteria strains compared to the solvent control, in both the presence and absence of metabolic activation.

The test item showed no relevant or dose-related increase in the number of revertants in the Salmonella typhimurium test strains TA98, TA100, TA102, TA1535 and TA1537 in all evaluated experiments.