Phosphonic acid, P-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-, sodium salt (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Arrival of the Test Item: 16 June 2011 Date of Final Report: 24 February 2012

Reliability:

1 (reliable without restriction)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 487

Deviations:

yes

Remarks:

See report for details. deviation did not affect quality of the study

Principles of method if other than guideline:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 487, ‘’In Vitro Mammalian Cell Micronucleus Test’’, adopted 22nd July, 2010.

Kirsch-Volders M. et al., “Report from in vitro micronucleus assay working group”, (2003), Mut. Res. 540, 153 – 163.

Kirsch-Volders M. et al., “Report from the in vitro micronucleus assay working group”, (2000), Environm. Mol. Mut. 35, 167 - 172.

Von der Rude W. et al, “In vitro micronucleus assay with Chinese hamster V79 cells results of a collaborative study with in situ exposure to 26 chemical substances”, (2000), Mut. Res, 468, 137- 163.

Fenech M., “Cytokinesis-block micronucleus cytome assay”, (2007), Nature Protocols 2 (5), 1084-1104.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

sodium hydrogen (3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)phosphonate

EC Number:

700-812-1

Cas Number:

1189052-95-6

Molecular formula:

C8H5F13O3PNa

IUPAC Name:

sodium hydrogen (3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)phosphonate

Details on test material:

Name: FHP-OHS
Batch No.: Z1601

Component 1:
Chemical Name: 3,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluorooctyl-
phosphonic acid sodium salt (1:1)
CAS No.: 1189052-95-6
Formula: C8H5F13O3P*Na
Purity: 93%

Component 2:
Chemical Name: 3 ,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluorooctyl-
phosphonic acid
CAS No.: 252237-40-4
Formula: C8H6F13O3P
Purity: 7%

Date of Manufacture: 9 June 2011
Expiration Date: 3 years
Colour: white to yellow
Storage Conditions: at room temperature
Date of Analysis: 10 June 2011 (cf. Certificate of Analysis attached
in the appendix of attached report)
Safety Precautions: Routine hygienic procedures were sufficient to assure personnel health and safety.

Method

Target gene:

See attached report

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Exposure Concentrations: The selection of the test item concentrations used and evaluated in the experiments based on cytotoxicity of the test item that intensified with increasing test item concentrations and occurred at concentrations where a low and constant level of precipitation was observed.

Duplicate cultures were treated at each concentration. The following concentrations were used in the main experiments:

Experiment I:
without metabolic activation: 750, 1000, 1200, 1300, 1400, 1500, 1550, 1600, 1650 and 1700 µg/mL
with metabolic activation: 1500, 1600, 1650, 1675, 1700, 1725, 1750, 1775 and 1800 µg/mL

Experiment II:
without metabolic activation: 31.6, 100, 316, 1000, 1250, 1500, 1600, 1700, 1800, 1900, 2000, 2250 and 2500 µg/mL

The following concentrations were selected for the microscopic analyses:

Experiment I with short exposure (4 h):
without metabolic activation: 1200, 1300, 1400 and 1500 µg/mL with metabolic activation: 1500, 1600, 1650 and 1675 µg/mL

Experiment II with extended exposure (20 h):
without metabolic activation: 1250, 1500, 1600 and 1700 µg/mL

Vehicle / solvent:

MEM

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Colecimide

Details on test system and experimental conditions:

Test System

Cells: V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens, These cells are chosen because of their stable karyotype and their low spontaneous induction rate of micronucleus formation under standardized culture conditions. These facts are necessary for the appropriate performance of the study.

The V79 cells (ATCC, CCL-93) are stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking of mycoplasma infections were carried out before freezing.

For the experiments thawed cultures are set up in 75 cm2 cell culture plastic flasks at 37°C in a 5% carbon dioxide atmosphere (95% air). 5 x 105 cells per flask are seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FBS (foetal bovine serum) and subcultures are made every 3-4 days.

Mammalian Microsomal Fraction S9 Homogenate: An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary. Many substances only develop mutagenic potential when they are metabolized by the mammalian organism. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).

The S9 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and B-naphthoflavone (100 mg/kg bw) [10,11] for three consecutive days by oral route.

The following quality control determinations are performed:
a) Biological activity in:
- the Salmonella typhimurium assay using 2-aminoanthracene
- the mouse lymphoma assay using benzo[a]pyrene
- the chromosome aberration assay using cyclophosphamide.
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4.5 mL and stored at ≤-75°C.
The protein concentration in the S9 preparation (Lot: 210711) was 38.5 mg/mL.

S9 Mix: An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.

Culture Medium:
Complete Culture Medium: MEM medium supplemented with:
10 % foetal bovine serum (FBS)
100 U/100 µg/mL penicillin/streptomycin solution
2 mM L-glutamine
2.5 µg/mL amphotericin
25 µM HEPES

Treatment Medium (short-time exposure): Complete culture medium without FBS.

After Treatment Medium / Treatment Medium (long-time exposure): Complete culture medium with 10% FBS and 1.5 µg/mL cytochalasin B.

(10). Elliot, EM., Combes, RD., Elcombe, CR., Gatehouse, DO., Gibson, G.G., Mackay, J.M. and Wolf, R.C. (1992). Report of UK Environmental Mutagen Society Working Party. Alternatives to Aroclor 1254- induced S9 in In Vitro Metabolic Activation in Mutagenesis Testing Mutagenesis, 7, 175-177

(11). Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T. (1976). A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. In: de Serres, F.)., Fouts, JR., Bend, J.R. and Philpot R.M., (eds). In Vitro Metabolic Activation in Mutagenesis Testing Elsevier, North-Holland, 85-88

Evaluation criteria:

See Attached report

Statistics:

Not used in the analysis

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Additional information on results:

See attached report

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Pre-Experiment for Toxicity: According to OECD guideline 487 the highest recommended dose is 5000µg/mL. The test item was dissolved in MEM medium and treated with ultrasound for 15 minutes at 37°C. Precipitation of the test item was noted at a low and constant level at concentrations of 500µg/mL and higher (without metabolic activation) and at concentrations of 31.3µg/mL and higher (with metabolic activation) at the end of treatment. The highest dose group evaluated in the pre-experiment was 1000µg/mL. The cytokinesis block proliferation index (CBPI) was used as parameter for toxicity. The concentrations evaluated in the main experiment based on the results obtained in the pre-experiment (Table 1 of attached report).   Precipitation: The test item was prepared in culture medium (MEM medium). A low level of precipitation was observed at all concentrations used and evaluated in experiment I and II that did not intensify with increasing test item concentrations.   Toxicity: In experiment I without metabolic activation no decrease of the relative CBPI below 70 % was noted up to a concentration of 1400µg/ml. At a concentration of 1500µg/mL a relative CBPI of 57% was observed. In experiment I with metabolic activation no decrease of the relative CBPI below 70 % was noted up to a concentration of 1600µg/ml. At a concentration of 1650µg/mL a relative CBPI of 66% and at a concentration of 1675µg/mL a relative CBPI of 52% was noted.   In experiment II without metabolic activation no decrease of the relative CBPI below 70% was noted up to a concentration of 1500µg/mL. At a concentration of 1600µg/mL a relative CBPI of 67% and at a concentration of 1700µg/mL a relative CBPI of 48% was noted.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the study described and under the experimental conditions reported, FHP-OHS did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.

Therefore, FHP-OHS is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Executive summary:

Summary Results: In order to investigate FHP-OHS for a possible potential to induce micronuclei in Chinese hamster V79 cells an in vitro a micronucleus assay was carried out.   The test item was dissolved in cell culture medium, treated with ultrasound for 15 minutes at 37°C and diluted within 1 hour prior to treatment. The pH-value detected with the test item was within the physiological range (7.0-7.4).   The selection of the concentrations used in experiment I and II was based on data from the pre-experiment according to the guidelines.   In experiment I 1500µg/mL without metabolic activation and 1675µg/mL with metabolic activation was selected as highest dose group for the microscopic analysis of micronuclei.   In experiment II without metabolic activation 1700µg/mL was selected as highest dose group for the microscopic analysis of micronuclei.   The following concentrations were evaluated: Experiment I with short exposure (4 h): without metabolic activation: 1200, 1300, 1400 and 1500µg/mL with metabolic activation: 1500, 1600, 1650 and 1675µg/mL   Experiment II with extended exposure (20 h): without metabolic activation: 1250,1500, 1600 and 1700µg/mL   The test item was prepared in culture medium (MEM medium). A low level of precipitation was observed at all concentrations used and evaluated in experiment I and II that did not intensify with increasing test item concentrations.   In experiment I without metabolic activation no decrease of the relative CBPI below 70 % was noted up to a concentration of 1400µg /ml. At a concentration of 1500µg /mL a relative CBPI of 57% was observed. In experiment I with metabolic activation no decrease of the relative CBPI below 70 % was noted up to a concentration of 1600µg/ml. At a concentration of 1650µg/mL a relative CBPI of 66% and at a concentration of 1675µg/mL a relative CBPI of 52% was noted. In experiment II without metabolic activation no decrease of the relative CBPI below 70% was noted up to a concentration of 1500µg/mL. At a concentration of 1600µg/mL a relative CBPI of 67% and at a concentration of 1700µg/mL a relative CBPI of 48% was noted.   The micronucleus frequencies found in the groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control.   The nonparametric x2 Test was performed to verify the results in both experiments. No statistically significant enhancement (p<0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I and II without metabolic activation (Table 9-11 of attached report). In experiment I with metabolic activation the micronucleus frequencies at concentrations of 1600µg/mL and 1675µg/mL were statistically significantly increased compared to the corresponding negative control (as determined by the nonparametric x2 Test), However, these micronucleus frequencies were within the range of the historical negative control data and no dose-response relationship could be observed. Based on this data the observed increases were regarded as not biologically relevant.   Ethylmethanesulfonate (, 900 and 600µg/mL) and Cyclophosphamide (CPA, 2.5µg/mL) were used as clastogenic controls. Colcemide (0.08 and 0.8µg/mL) was used as aneugenic control. All induced distinct and biologically relevant increases of micronucleus frequency. This demonstrates the validity of the assay.