Phosphoric acid, mono- and di-C16-18-alkyl esters

Genetic toxicity: in vitro

Administrative data

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:

April - August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: USA, EPA (TSCA) OCSPP harmonized guidelines 40 CFR 799.9510 TSCA bacterial reverse mutation test

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:

Octadecyl dihydrogen phosphate

EC Number:

220-983-1

EC Name:

Octadecyl dihydrogen phosphate

Cas Number:

2958-09-0

Molecular formula:

C18H39O4P

IUPAC Name:

octadecyl dihydrogen phosphate

Constituent 2

Reference substance name:

Hexadecyl dihydrogen phosphate

EC Number:

222-581-1

EC Name:

Hexadecyl dihydrogen phosphate

Cas Number:

3539-43-3

Molecular formula:

C16H35O4P

IUPAC Name:

hexadecyl dihydrogen phosphate

Constituent 3

Reference substance name:

Dioctadecyl hydrogen phosphate

EC Number:

221-237-8

EC Name:

Dioctadecyl hydrogen phosphate

Cas Number:

3037-89-6

Molecular formula:

C36H75O4P

IUPAC Name:

dioctadecyl hydrogen phosphate

Constituent 4

Reference substance name:

Dihexadecyl hydrogen phosphate

EC Number:

218-594-7

EC Name:

Dihexadecyl hydrogen phosphate

Cas Number:

2197-63-9

Molecular formula:

C32H67O4P

IUPAC Name:

dihexadecyl hydrogen phosphate

Constituent 5

Reference substance name:

hexadecyl octadecyl hydrogen phosphate

Cas Number:

93803-11-3

Molecular formula:

C34H71O4P

IUPAC Name:

hexadecyl octadecyl hydrogen phosphate

impurity 1

Reference substance name:

Unidentified impurities, likely the C16 and C18, and mixed C16/C18 trialkyl phosphates based 31P NMR chemical shift.

Molecular formula:

N/A

IUPAC Name:

Unidentified impurities, likely the C16 and C18, and mixed C16/C18 trialkyl phosphates based 31P NMR chemical shift.

Test material form:

solid

Details on test material:

CAS Number 39471-52-8

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

Experiment 1: Concentrations of 1.5, 5, 15, 50, 150, 500, 1500 & 5000 µg/plate.
Experiment 2: Concentrations of 50, 150, 500, 1500 & 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:Tetrahydrofuran
Justification: The test material was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but fully soluble in tetrahydrofuran at 200 mg/mL in solubility checks performed in-house within the testing laboratory. Tetrahydrofuran was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Remarks:

ENNG: 2µg/plate for WP2uvrA, 3µg/plate for TA100, 5µg/plate for TA1535; 9AA: 80µg/plate for TA1537; 4NQO: 0.2µg/plate for TA98.

Negative solvent / vehicle controls:

yes

Remarks:

Tetrohydrofuran

True negative controls:

not specified

Positive controls:

yes

Remarks:

2AA: 1µg/plate for TA100, 2µg/plate for TA1535 and TA1537, 10µg/plate for WP2uvrA; BP: 5µg/plate for TA98.

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The test material was accurately weighed and approximate half-lg dilutions prepared in tetrahydrofuran by mixing on a vortex mixer and sonication for 30 minutes at 40°C in the day of each experiment. Tetrahydrofuran is toxic to the bacterial cells at above 50µl after employing the pre-incubation modification, therefore all of the formulation for Experiment 2 were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.025 mL (25µL) aliquots. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneiry and stability of the test material formulation is not a requirement of the test guidelines and was, therefore not determined. This is an exeption with regards to GLP and has been reflected in the GLP compliance statement. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2mm sodium alumino-silicate pellets with a nominal pore diameter of 4E-4 microns.

TEST PROCEDURE
Experiment 1 - Plate Incorporation Method
- Dose: The maximum concentration was 5000µg/plate. Eight concentrations of the test material (1.5, 5, 15, 50, 150, 500, 1500 amd 5000 µg/plate) were assayed in triplicate against each tester strain.
- Without activation: 0.025mL of the concentration of test material or vehicle or 0.1 mL of appropriate positive control was added to 2mL of trace amino-acid supplemented media containing 0.1mL of one of the bacterial strain cultures and 0.5mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Each concentration of test material, positive control and each bacterial strain, was assayed using triplicates plates.
- With activation: The procedure was as above, except that following the addition of the test material formulation and bacterial culture, 0.5mL of S-9 mix was added to the trace amino-acid supplemented media in place of phosphate buffer.

Experiment 2 - Pre-Incubation Method
- Dose: The dose range used for Experiment 2 was determined by the results of Experiment 1, 50, 150, 500, 1500 amd 5000 µg/plate.
- Witout activation: 0.1mL of the appropriate bacterial strain culture, 0.5mL of phosphate buffer and 0.025mL of the test material formulation or vehicle or 0.1mL of appropriate positive control were incubated at 37 ± 3°C for 20 minutes (with shaking) prior to addition of 2mL of aminio-acid supplemented media and subsequent plating onto Vogel-Bonner plates.
-With activation: The procedure was as above, except that following the addition of the test material formulation and bacterial strain culture, 0.5mL of S-9 mix was added to the tube instead of phosphate buffer. All testing for this experiment was performed in triplicate.
All plates were incubated at 37 ± 3°C for 20 minutes for approximately 48 hours.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

The test material was considered to be non-mutagenic under the conditions of the study.