Triisodecyl phosphite

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

29st July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Triisotridecyl phosphite

EC Number:

278-758-9

EC Name:

Triisotridecyl phosphite

Cas Number:

77745-66-5

Molecular formula:

C39H81O3P

IUPAC Name:

triisotridecyl phosphite

Test material form:

liquid

Specific details on test material used for the study:

NAME OF TEST MATERIAL (as stated in the study report): Trisisotridecylphosphite

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Valtris Specialty Chemicals, Batch No. 330965
- Expiration date of the lot/batch: March 02, 2019
- Purity: 100% (UVCB substance)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in original container.
- Stability under test conditions: Phosphites have the potential to hydrolyze in water
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in DMSO at 100 µL/mL.

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Japanese Collection of Research Bioresources

The cell line free from mycoplasma contamination was used in the study. Cultures were free from any contamination during the conduct of the study. CHO-K1 cell line passage number 39 and 38 was used in cytotoxicity test and in main study respectively.

MEDIA USED
α-MEM (Minimum Essential Medium, Eagle α-Modification with nucleosides) with nucleosides (Gupta R.S., 1984) with 10% heat inactivated, sterile, fetal bovine serum was used as the culture medium to grow the CHO-K1 cell line. Culture medium was supplemented with antibiotic and antimycotic solution (Penicillin: 50 IU/mL; Streptomycin: 50 µg/mL and Amphotericin B: 0.25-0.5 μg/mL). At the time of selection Minimum Essential Medium Eagle -modification without nucleosides (-MEM w/o NS) with 10% dialyzed fetal bovine serum was used.

The medium to eliminate the existing mutants in the culture for treatment was prepared by addition of 2 mL of reconstituted HAT supplement to 98 mL of α-MEM w/o NS with 5% fetal bovine serum [50X vial of HAT media supplement was reconstituted using 10 mL of sterile α-MEM w/o NS. The reconstituted supplement contains 5 x 10-5 Hypoxanthine, 2 x 10-5 M Aminopterine and 8 x 10-4 M Thymidine].

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction (treated with Aroclor 1254)

Test concentrations with justification for top dose:

0.006, 0.013, 0.025, 0.05, 0.1 and 0.2 µL/mL in the absence and 0.031, 0.063, 0.125, 0.25, 0.5 and 1 µL/mL in the presence of metabolic activation. Based on the results of solubility, precipitation and pH tests, 1 µL/mL was selected as the highest concentration to be tested.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility testing

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 -9 days
- Selection time (if incubation with a selection agent): 8 days

NUMBER OF REPLICATIONS: 4 flasks for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

Guidelines and pretest results

Evaluation criteria:

Per guidelines

Statistics:

Weighted regression analysis was performed to evaluate the dose response relationship (Li, A.P. et al., 1987; Hsie, A.W. et al., 1981) on treatment groups against the negative control group (excluding positive controls).

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No significant dose-related increase in the mutation frequency was observed in any of the treatment concentrations between 0.006 to 0.2 µL/mL of culture medium in absence or 0.031 to 1 µL/mL of culture medium in presence of metabolic activation (2% v/v S9 mix), and the induced mutation frequencies were comparable to that of the negative control group. All negative controls were within the historical control limits and positive controls showed an increase in the mutant frequency. No relevant influence of the test item on pH value or osmolality was observed in the absence or presence of metabolic activation.

Doses were selected based on a cytotoxicity test.

All criteria for a valid study were met.

Applicant's summary and conclusion

Conclusions:

Under these guideline test conditions, it is concluded that trisisotridecylphosphite does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells, in the absence and presence of metabolic activation.