Reaction products of triphenyl phosphite and isodecanol (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Triphenyl phosphite

EC Number:

202-908-4

EC Name:

Triphenyl phosphite

Cas Number:

101-02-0

Molecular formula:

C18H15O3P

IUPAC Name:

triphenyl phosphite

Constituent 2

Reference substance name:

Triphenyl phosphate

EC Number:

204-112-2

EC Name:

Triphenyl phosphate

Cas Number:

115-86-6

Molecular formula:

C18-H15-O4-P

IUPAC Name:

triphenyl phosphate

Specific details on test material used for the study:

TEST MATERIAL: Triphenyl Phosphite
- Purity: 99.48%

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: UPL Limited, Jhagadia, India. Batch No. CO05TPI078
- Expiration date of the lot/batch: February 2018
- Purity test date: March 29, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature in original container
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in DMSO
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No significant changes in pH (±1 unit) or osmolality (≥ 50 mOsm/kg H2O) were observed at concentrations up to 2000 µg/mL of culture medium. In addition, no precipitation was observed up to the test concentration of 1000 µg/mL while turbidity was observed at concentration of 2000 µg/mL of culture medium.

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: human volunteer
- Sex, age and number of blood donors if applicable: 28 year old healthy male
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Methods for maintenance in cell culture if applicable: The whole blood was cultured in RPMI-1640 (Roswell Park Memorial Institute) with
L-glutamine and 25 mm HEPES (Sigma R4130) containing antibiotic and antimycotic solution (penicillin: 50 IU/mL; streptomycin: 50 µg/mL and amphotericin B: 0.25 μg/mL) supplemented with 20% heat-inactivated (56 °C; 30 min.) fetal bovine serum.

Cultures were prepared separately in centrifuge tube by adding heparin sodium containing 0.5 mL of whole blood into 9.5 mL of complete medium [containing 20% heat-inactivated (56 °C; 30 min.) fetal bovine serum and 2% Phytohaemagglutinin (PHA-M)] in culture tubes and incubated at
37 ± 1 °C and 5% CO2 in a CO2 incubator for approximately 48 hours.

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction (treated with Aroclor 1254)

Test concentrations with justification for top dose:

Phase I: concentrations of 3.75, 7.5, 15, 30 and 60 µg triphenyl phosphite/mL of culture for approximately 4 h in the absence and 5, 10, 20, 40 and 80 µg triphenyl phosphite/mL of culture medium in the presence (2% v/v S9) of a metabolic activation.

Phase II: concentrations of 5, 10, 20, 40 and 80 µg triphenyl phosphite/mL of culture (supplemented with 10% FBS), for approximately 24 h without S9 in the absence of metabolic activation with cytochalasin B (6 µg/mL).

Test concentrations were selected based on results of preliminary cytotoxicity screening study (55 +/- 5% inhibition of replicative index)

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Vinblastine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Phase I - 4 hrs; Phase II - 24 hrs

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Slides were prepared from each culture tube by pouring approximately about 0.5 mL of the fixed cell suspension, drop by drop on two, pre-cleaned, ice-chilled slides. The slides were dried over a slide warmer and labelled with study number, treatment code, absence/presence metabolic activation and slide number. The dried slides were stained with 5% Giemsa in phosphate buffer for 30 minutes. The slides were made permanent by mounting a cover slip with DPX mountant. All slides were coded before microscopic scoring and decoded after scoring.

NUMBER OF CELLS EVALUATED: A minimum of 2000 binucleated cells

DETERMINATION OF CYTOTOXICITY
- Method: replicative index

Rationale for test conditions:

test guideline

Evaluation criteria:

Assay acceptance: valid negative (untreated), solvent and positive controls; appropriate levels of cytotoxicity (replicative index); adequate number of cells and concentrations analysable; Assay evaluation: statistical significance; biological significance; confounders (changes in pH, osmolality); historical control range.

Statistics:

Data on micronuclei containing binucleated cells were subjected to Shapiro-Wilk’s test for normality and Bartlett’s test to assess homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test (Gad and Weil, 1994). Where the data did not meet suitable homogeneity of variance, Student's t-test was performed to determine the level of significance between vehicle control, three selected test concentrations (selected based on the replicative index data) and positive controls.

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Applicant's summary and conclusion

Conclusions:

In a reliable OECD Guideline 487 GLP study, TPP did not show any potential to induce micronuclei or clastogenic or aneugenic potential in cultured human peripheral blood lymphocytes, both in the absence and presence of metabolic activation system, at concentrations up to those producing recommended levels of cytotoxicity (55 ± 5% inhibition of replicative index).