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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Arrival of the Test Item: 16 June 2011 Date of Draft Study Plan: 27 June 2011 Date of Final Study Plan: 28 June 2011 Start of Experiment: 29 June 2011 End of Experiment: 01 July 2011 Date of Draft Report: 08 July 2011 Date of Final Report: 21 July 2011

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Principles of method if other than guideline:

Regulation 440/2008, Method 1140: “Skin Corrosion”, May 30, 2008 (1).

OECD (2002). OECD Guideline for the Testing of Chemicals. No. 431: In Vitro Skin Corrosion: Human Skin Model Test. 13 April 2004 (2).

(1) Council Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Official Journal of the European Communities L142, 394-399.

(2) OECD (2002). OECD Guideline for the Testing of Chemicals. No. 431: In Vitro Skin Corrosion: Human Skin Model Test. 13 April 2004.

GLP compliance:

yes (incl. QA statement)

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
- Safety Precautions: Routine hygienic procedures were
- sufficient to assure personnel health and
safety

Test animals

Species:

other: EpiDermTM Skin Model was used

Strain:

other: in vitor study so N/A

Details on test animals or test system and environmental conditions:

N/A

Test system

Type of coverage:

open

Preparation of test site:

other: invitro so no requirement

Vehicle:

water

Controls:

not required

Amount / concentration applied:

Dose Groups:
1. Negative control 50 µL distilled water
2. Positive control 50 µL 8 N KOH
3. Test Item 25mg + 25 µL H20
The test was performed on a total of 4 tissues per dose group, 2 replicates for each treatment period (3 min. and 60 min. exposure time).

Duration of treatment / exposure:

The test was performed on a total of 4 tissues per dose group, 2 replicates for each treatment period (3 min. and 60 min. exposure time)

Observation period:

60 mins

Number of animals:

N/A

Details on study design:

Test System: The test was carried out with the reconstituted three-dimensional human skin model EpiDermTM (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell®). The EpiDermTM skin model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous, granular and cornified layers analogous to those found in vivo.

Pre-Experiment: To check the MTT-reducing capability of the test item 30 mg of the test item were mixed with 1 mL MTT medium and incubated for 1 h at room temperature. If the mixture turns blue/purple, the test item is presumed to have reduced MTT. This check can only be used to explain unexpected results, but it can not be used for quantitative correction of results.

Experimental Procedure: Upon receipt of the EpiDermTM - kit, the tissues were transferred into 6-well plates containing 900 µL prewarmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37 ± 1°C, 5.0% CO2 / 95% air for at least 1 h. Then the medium was replaced by 900 RL fresh assay medium. The 6-well plate used for the 3 mm. experiment was placed back into the incubator. The other plate was used for the 60 mi treatment. About 1 h before the end of the first treatment period the MTT solution was prepared by mixing the MTT concentrate with the MTT diluent and pre-warmed in the incubator.

60 min. experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the 6-well plate was incubated at 37 ± 1°C, 5.0% CO2 / 95% air.

3 min. experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 sec. was kept between dosing. After 3 mm. of application, with foreceps, the first insert was removed from the 6-well plate. Using a wash bottle the tissue was gently rinsed about 20 times with PBS (phospate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate” containing 300 RL prewarmed assay medium per well. All inserts were treated in the same manner. Then the inserts were transferred into a prepared 24-well “MTT assay plate” containing 300 µL prewarmed MTT solution, The plate was incubated for 3 h at 37 ± 1 °C, 5.0% CO2 / 95% air.

60 min. experiment: after 60 min. application, with foreceps, the first insert was removed from the 6-well plate. Using a wash bottle the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate” containing 300 µL prewarmed assay medium per well. All inserts were treated in the same manner. Then the inserts were transferred into a prepared 24-well “MTT assay plate” containing 300 µL prewarmed MTT solution. The plate was incubated for 3 h at 37 ± 1°C, 5.0% CO2 / 95% air.

3 min. and 60 min. experiment: after the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. Then the inserts were transferred into new 24-well “extraction plates”. 2 mL of isopropanol were pipetted into each insert, thus the insert was covered from both sides. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out over night without shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min.
Per each tissue 3 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 550 nm without reference wavelength in a plate spectrophotometer.

Results and discussion

In vivo

Irritant / corrosive response data:

See Attahced repot

Other effects:

none

Any other information on results incl. tables

Pre-Experiment:The mixture of 100 µL / 30mg test item per 1 ml MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. For table see attached report  * mean OD550≥ 0.8 ** mean relative tissue viability of the 3 min. positive control ≤30% *** inter tissue viability difference ≤ 30%  for table see attaced report * mean OD550≥ 0.8 *** inter tissue viability difference ≤ 30%   Historical data:   For table see attached report   The potential of the test item to induce skin corrosion was analysed by using the three-dimensional human skin model EpiDerm^TM, comprising a reconstructed epidermis with a functional stratum corneum.   In the present study FHP-OHS was applied topically to the EpiDerm^TMtissue for 3 min. and 60 min. followed by immediate determination of cytotoxic effects via MTT reduction assay.   Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods compared to the corresponding negative control tissues concurrently treated with A. dest.   The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥ 50% (88%) after 3 mm. treatment and ≥ 15% (83%) after 60 min. treatment.   The controls confirmed the validity of the study. The mean 0D550of the two negative control tissues was ≥ 0.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was ≤ 30% (12%) after 3 min. treatment. The maximum inter tissue difference of replicate tissues of all dose groups was ≤ 30% (1.3% - 14.8%).

Applicant's summary and conclusion

Interpretation of results:

other: non corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

In this study under the given conditions the test item showed no corrosive effects. The test item is classified as “non corrosive”.

Executive summary:

In the present study the skin corrosivity potential of FHP-OHS was analysed. Since corrosive chemicals are cytotoxic after a short time exposure to the stratum corneum of the epidermis the cytotoxic effects of the test item on EpiDerm^TM, a reconstituted three-dimensional human epidermis model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 min. and 60 min. exposure period and compared to those of the concurrent negative controls.   In this study under the given conditions the test item showed no corrosive effects. The relative mean tissue viability after 3 min. treatment was ≥ 50% and after 60 min. treatment ≥ 15%. The test item is therefore classified as “non corrosive”.