Triglycerides, C16-C18 (even numbered) and C18 (unsaturated), maleated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: negative (OECD 471) Glycerol: - Bacterial reverse mutation assay: negative (similar to OECD 471) - In vitro mammalian chromosome aberration test: negative (OECD 473) - Sister chromatid exchange assay in mammalian cells: negative (similar to OECD 476) Fatty acids, C14-18 and C16-18-unsatd., maleated: - - Bacterial reverse mutation assay: negative (OECD 471) - Mammalian cell gene mutation assay: negative (OECD 476) - In vitro mammalian chromosome aberration test: negative (OECD 473)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-11-07 to 2014-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

His

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver microsomal fraction

Test concentrations with justification for top dose:

Experiment I:
10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment II:
10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(for TA 98 and TA 102 with and without metabolic activation and for TA 100, TA 1535 and TA 1537 with metabolic activation)
1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (for TA 100, TA 1535 and TA 1537 without metabolic activation activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

A. dest., BSL Lot No. 141031

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, Lot No. 14I013997

Positive controls:

yes

Positive control substance:

other:

Remarks:

Positive control without metabolic activation: sodium azide for tester strains TA100, TA1535; 4-nitro-o-phenylene-diamine for TA98, TA1537; methylmethanesulfonate for TA102. Positive control with metabolic activation: 2-aminoanthracene for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
-For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


- Preincubation period: 60 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.



NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic 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 dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Additional information on results:

No precipitation of the test item was observed in any tester strain used in experiment I (with and without metabolic activation). Precipitation was observed in all tester strains used in experiment II (with and without metabolic activation).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

In experiment I toxic effects of the test item were observed in tester strains TA 100, TA 1535 and TA 1537 at a concentration of 5000 µg/plate (without metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.4 found in tester strains TA 1535 and TA 1537 at a concentration of 1000 µg/plate (without metabolic activation) and additionally down to a mutation factor of 0.3 in tester strain TA 1535 at a concentration of 316 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.

In experiment II toxic effects of the test item were noted in tester strains TA 100 and TA 1537 at concentrations of 100 µg/plate and higher (without metabolic activation). In tester strain TA 1535 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.4 found in tester strain TA 1537 at a concentration of 3.16 µg/plate, 31.6 µg/plate and 316 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, MSOHO did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, MSOHO is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to MSOHO at concentrations of 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (Experiment I) and 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (for TA 98 and TA 102withandwithoutmetabolic activation and for TA 100, TA 1535 and TA 1537withmetabolic activation at Experiment II) and 1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

(for TA 100, TA 1535 and TA 1537withoutmetabolic activation activation at experiment II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II). MSOHO was

tested up to the limit concentration of 5000 µg/plate in all tester strains used.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Genetic toxicity of High oleic sunflower oil, maleated in bacteria was evaluated in a study performed according to OECD 471 and GLP, where bacteria strains Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 were incubated with doses of 1 to 5000 µg/plate of the substance using the plate incorporation test (experiment I) and the pre-incubation test (experiment II), both with and without metabolic activation, respectively. Although some cytotoxic effects have been found in experiment I at the highest dose tested without metabolic activation, while in experiment II cytotoxicity was observed at concentration of 100 µg/plate and higher without metabolic activation, no increase in number of revertants was noted in all tester strains and all doses tested in both experiments. Thus, the test substance is considered to be non-mutagenic in the bacterial reverse mutation assay..

Glycerol was tested in an Ames test, where S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1530 were treated with concentrations up to 1000 µg/plate with an without metabolic activation. Since no increase in the number of revertants was found, glycerol was found to be non-mutagenic in the bacterial reverse mutation assay.

Glycerol was assessed in a chromosomal aberration assay conducted comparable to OECD guideline 473 where it was tested in Chinese hamster Ovary Cells with concentrations up to 1000 mg/mL with and without metabolic activation. In the same study, glycerol was also assessed in a sister chromatide exchanges assay conducted comparable to OECD guideline 476 where it was tested in Chinese hamster Ovary Cells with concentrations up to 1000 mg/mL with and without metabolic activation. As a result of both tests, no mutagenic response was noted.

Fatty acids, C14-18 and C16-18-unsatd., maleated was tested in a mammalian cell gene mutation assay performed according to GLP and OECD 476. Mouse lymphoma L5178Y cells were treated as duplicates in each of three experiments with concentrations up to 5000 µg/mL for 3 h exposure (–/+S9) and 24 h exposure (–S9) (experiment 1), for 3 h exposure with (+S9) and 24 h exposure without metabolic activation (–S9) (experiment 2) and for 24 h exposure without metabolic activation (–S9) (experiment 3, respectively. As result of all experiments, no genotoxicity was found.

Fatty acids, C14-18 and C16-18-unsatd. maleated was also tested for the potential to induce chromosome aberration in a study performed in accordance to OECD 473, where Chinese hamster lung fibroblasts (V79) were treated with concentrations up to up to 5000 µg/mL with and without metabolic activation. No repeatable dose-dependent induction of chromosome aberrations in Chinese hamster V79 cells was observed so that the substance was considered not clastogenic under the condition of the test.

Fatty acids, C14-18 and C16-18-unsatd. maleated was additionally found to be negative in an Ames test performed in accordance with OECD 471 and GLP. Bacteria strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 were treated with concentrations up to 5000 μg/plate with and without metabolic activation. Although some precipitation at the highest dose in all strains and some cytotoxicity has been noted, no increase in number revertants was found.

The overall results on genetic toxicity testing with High oleic sunflower oil, maleated and the source substances Fatty acids, C14-18 and C16-18-unsatd., maleated and glycerol clearly show that High oleic sunflower oil, maleated can be regarded as not mutagenic.

Justification for selection of genetic toxicity endpoint
Study according or comparable to OECD guidelines

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.