Fatty acids, C18-unsatd., phosphates

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:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

In the pre - incubation assay in the tester strain TA100, the test item solutions were incubated for 29 minutes. The deviation had no effect on the study integrity.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

9-(dimethoxyphosphoryl)octadecanoic acid 10-(dimethoxyphosphoryl)octadecanoic acid

Molecular formula:

C20H41O5P

IUPAC Name:

9-(dimethoxyphosphoryl)octadecanoic acid 10-(dimethoxyphosphoryl)octadecanoic acid

Constituent 2

Reference substance name:

9,12-bis(dimethoxyphosphoryl)octadecanoic acid 9,13-bis(dimethoxyphosphoryl)octadecanoic acid 10,12-bis(dimethoxyphosphoryl)octadecanoic acid 10,13-bis(dimethoxyphosphoryl)octadecanoic acid

Molecular formula:

C22H46O8P2

IUPAC Name:

9,12-bis(dimethoxyphosphoryl)octadecanoic acid 9,13-bis(dimethoxyphosphoryl)octadecanoic acid 10,12-bis(dimethoxyphosphoryl)octadecanoic acid 10,13-bis(dimethoxyphosphoryl)octadecanoic acid

Constituent 3

Reference substance name:

Oleic acid

EC Number:

204-007-1

EC Name:

Oleic acid

Cas Number:

112-80-1

Molecular formula:

C18H34O2

IUPAC Name:

octadec-9-enoic acid

Constituent 4

Reference substance name:

Linoleic acid

EC Number:

200-470-9

EC Name:

Linoleic acid

Cas Number:

60-33-3

Molecular formula:

C18H32O2

IUPAC Name:

octadeca-9,12-dienoic acid

Constituent 5

Reference substance name:

Dimethyl (2-oxopropyl)phosphonate

EC Number:

224-110-5

EC Name:

Dimethyl (2-oxopropyl)phosphonate

Cas Number:

4202-14-6

Molecular formula:

C5H11O4P

IUPAC Name:

dimethyl (2-oxopropyl)phosphonate

Constituent 6

Reference substance name:

Dimethyl propylphosphonate

EC Number:

242-555-3

EC Name:

Dimethyl propylphosphonate

Cas Number:

18755-43-6

Molecular formula:

C5H13O3P

IUPAC Name:

dimethyl propylphosphonate

Constituent 7

Reference substance name:

Dimethyl methylphosphonate

EC Number:

212-052-3

EC Name:

Dimethyl methylphosphonate

Cas Number:

756-79-6

Molecular formula:

C3H9O3P

IUPAC Name:

dimethyl methylphosphonate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0101891886
- Expiration date of the lot/batch: 02 November 2017
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Stable, maximum temperature: 60 °C
- Solubility and stability of the test substance in the solvent/vehicle: Dimethyl sulfoxide: Not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Dimethyl sulfoxide: Not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No correction was made for the purity/composition of the test compound. A solubility test was performed based on visual assessment. The test item was dissolved in dimethyl sulfoxide. Test item concentrations were used within 1.5 hours after preparation. Any residual volumes were discarded.
- Final dilution of a dissolved solid, stock liquid or gel: A solubility test was performed based on visual assessment. The test item was dissolved in dimethyl sulfoxide.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and the WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate.

Vehicle / solvent:

The vehicle of the test item was dimethyl sulfoxide (Merck, Darmstadt, Germany).

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO; Saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 30 minutes
- Exposure duration:48±4 h

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a)The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b)The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a)The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b)In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium 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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn, was observed in tester strain TA100 in the absence of S9-mix at the highest concentration (5000 ug/ml) tested.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The 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.
In conclusion, based on the results of this study it is concluded that Fatty acids, C18-unsatd., phosphates is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of Fatty acids, C18-unsatd., phosphates. and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli(E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 0101891886 of Fatty acids, C18-unsatd., phosphates. was a yellow liquid. The test item was dissolved in dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn, was observed in tester strain TA100 in the absence of S9-mix at the highest concentration tested.  In all other tester strains, the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The 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.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that Fatty acids, C18-unsatd., phosphates.is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.