Reaction mass of 4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol and benzyltriphenylphosphonium, salt with 4,4'-[2,2,2-trifluoro-1-(trifluoromethyl) ethylidene]bis[phenol] (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: Micronucleus induction

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 February 2018 to 19 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline 487, updated and adopted 26 September 2014

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes

Remarks:

Refer to main study report

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol

EC Number:

216-036-7

EC Name:

4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol

Cas Number:

1478-61-1

Molecular formula:

C15H10F6O2

IUPAC Name:

4,4'-(1,1,1,3,3,3-hexafluoropropane-2,2-diyl)diphenol

Constituent 2

Reference substance name:

Benzyltriphenylphosphonium, salt with 4,4'- [2,2,2- trifluoro-1- (trifluoromethyl)ethylidene]bis[phenol] (1:1)

Cas Number:

75768-65-9

IUPAC Name:

Benzyltriphenylphosphonium, salt with 4,4'- [2,2,2- trifluoro-1- (trifluoromethyl)ethylidene]bis[phenol] (1:1)

Details on test material:

- Density: 1.38 g/cm3

Specific details on test material used for the study:

Purity: > 98 %

Method

Target gene:

micronuclei

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED- Source of cells: Peripheral blood lymphocytes were obtained from a healthy non-smoking individual who had no recent history of radiotherapy, viral infection or the administration of drugs.- Suitability of cells: Use of HPBL has been demonstrated to be sensitive to the genotoxicity test for detection of micronuclei of a variety of chemicals- Sex, age and number of blood donors if applicable: Male/ 25 years old / One donor- Whether whole blood or separated lymphocytes were used if applicable: Whole blood- Methods for maintenance in cell culture if applicable: Peripheral blood lymphocytes were cultured in complete medium (RPMI 1640 containing 15% heat inactivated fetal bovine serum, 2mM L glutamine, 100 units penicillin and 100 µg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium with 2% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.MEDIA USED- Type and identity of media including CO2 concentration if applicable: Complete medium (RPMI 1640 containing 15% heat inactivated fetal bovine serum, 2mM L glutamine, 100 units penicillin and 100 µg/mL streptomycin) and RPMI 1640 Serum-free medium supplemented with 2 mM L glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin- Properly maintained: [yes/no]: Yes

Cytokinesis block (if used):

Cytochalasin B (cytoB) was dissolved in DMSO to a stock concentration of 2 mg/mL. It was used at 6 µg/mL concentration to block cytokinesis.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO - Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, upon sonication for five minutes at 36.8 ºC, the test substance formed a clear and soluble solution in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Vinblastine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION- Exposure duration: HPBL were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9, by incorporation of the test substance vehicle mixture into the treatment medium.
STAIN (for cytogenetic assays): acridine orange
NUMBER OF REPLICATIONS: Two
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and the slides were prepared immediately after harvest. To prepare slides, the cells were collected by centrifugation and the suspension of fixed cells was applied to glass microscope slides and air-dried.
NUMBER OF CELLS EVALUATED: A minimum of 2000 binucleated cells from each concentration (if possible, 1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:• the micronucleus should have the same staining characteristics as the main nucleus• the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)• the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus
DETERMINATION OF CYTOTOXICITY- Method: Cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control

Evaluation criteria:

The test substance was considered to have induced a positive response if • at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and• the increase was concentration-related (p ≤ 0.05), and• results were outside the 95% control limit of the historical negative control data.The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p

Results and discussion

Test results

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative

Remarks:

Bisphenol AF salt was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the assay described in this report, Bisphenol AF Salt was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.

Executive summary:

The test substance, Bisphenol AF Salt,was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO)was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 µg/mL, which was the limit dose for this assay. Cytotoxicity [ ≥50%cytokinesis-blocked proliferation index(CBPI) relative to the vehicle control] was observed at doses ≥ 60 µg/mL in the non‑activated and S9‑activated 4-hour exposure groups and at doses ≥ 20 µg/mL in the non‑activated 24‑hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 200 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 10 to 60 µg/mL for the non‑activated 4‑hour exposure group, from 20 to 50 µg/mL for the S9-activated 4-hour exposure group, and from 2.5 to 15 µg/mL for the non-activated 24-hour exposure group.

In the initial micronucleus assay, cytotoxicity ( ≥ 50% CBPI relative to the vehicle control) was observed at doses ≥ 50 µg/mL in the non‑activated 4-hour exposure group; at doses ≥ 35 µg/mL in the S9‑activated 4-hour exposure group; and at doses ≥ 8 µg/mL in the non‑activated 24-hour exposure group. The doses selected for evaluation of micronuclei were 20, 40, and 55 µg/mL for the non-activated 4-hour exposure group and 2.5, 6, and 9 µg/mL for the non‑activated 24-hour exposure group.

In the non-activated 4-hour exposure group, no significant or dose‑dependent increases in micronuclei induction were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the S9-activated 4-hour exposure group, due to lack of requisite cytotoxicity, the micronucleus assay was repeated at doses ranging from 10 to 37 µg/mL. In the non-activated 24-hour exposure group, due to lack of statistically significant induction of micronuclei in the positive control, the micronucleus assay was also repeated at doses ranging from 2.5 to 15 µg/mL.

In the repeat assay, cytotoxicity ( ≥ 50% CBPI relative to the vehicle control) was observed at doses ≥ 30 µg/mL in the S9‑activated 4-hour exposure group and at doses ≥ 8 µg/mL in the non‑activated 24-hour exposure group. The doses selected for evaluation of micronuclei were 10, 20, and 30 µg/mL for the S9-activated 4-hour exposure group; and 2.5, 5, and 8 µg/mL for the non‑activated 24-hour exposure group.

No significant or dose‑dependent increases in micronuclei induction were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicate Bisphenol AF salt was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.