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

Endpoint summary

Description of key information

In vitro study shows FHP-OHS to be non corrosive and non irritant to the skin but highly irritation to the eye. Category 1 under GHS CLP legisltaion

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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)

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)

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

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.

Irritant / corrosive response data:

See Attahced repot

Other effects:

none

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%).

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”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Arrival of the Test Item: 16 June 2011 Date of Final Report: 06 September 2011

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 437

Principles of method if other than guideline:

This study followed the procedures indicated by internal BSL BIOSERVICE SOPs and the following internationally accepted guidelines and recommendations:

OECD Guideline for the Testing of Chemicals, number 437 “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants” (adopted: 7 September 2009) [3]

EPA Health Effects Test Guidelines, OPPTS 870.1000 “Acute toxicity testing background”, EPA 712-C-02-l 89, December 2002 [4]

Ministry of Health and Welfare (MHW, Japan): Japanese Guidelines for Nonclinical Studies of Drugs Manual 1995, Yakuji-Nippo Co. Ltd., 1995 (unofficial translation) [5]

Japanese Ministry of Agriculture, Forestry and Fisheries (JMAF’F), Guidelines for Preparation of Study Results, Eye Irritation Studies, Guideline 2-1-5. Notification 12 NohSan No. 8147, as partly revised in 16-Shouan-9260, on March 2005. English translation by IAI:ACIS on 17 October 2005 [6]

[3] OECD Guideline for the Testing of Chemicals, number 437 “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants” (adopted: 07 September 2009

[4] EPA Acute Toxicity Testing - Background. Health Effects Test Guidelines, OPPTS 870.1000. United States, EPA 7l2-C-02-189, December 2002, United States, Environmental Protection Agency, Prevention, Pesticides and Toxic Substances (7101)

[5] Ministry of Health and Welfare (MHW, Japan): Japanese Guidelines for Nonclinical Studies of Drugs Manual 1995, Yakuji-Nippo Co. Ltd., 1995 (unofficial translation)

[6] Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), Guidelines for Preparation of Study Results, Eye Irritation Studies, Guideline 2-1-5. Notification 12 NohSan No. 8147, as partly revised in 16-Shouan-9260, on March 2005. English translation by lAI:ACIS on 17 October 2005

GLP compliance:

yes (incl. QA statement)

Species:

other: Bovine corneal opacity and permeability assay

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Preparation of the Corneas:

The assay uses isolated corneas obtained as a by-product from an abattoir from freshly slaughtered animals.

The eyes were carefully examined for defects and any defective eyes were discarded.

The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Corneas were then mounted in corneal holders (MC2, Clermont, France) with the endothelial side against the 0-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1°C in a water bath.

Treatment of the Corneas:

After the incubation period, the medium was removed from both chambers and replaced with fresh Complete RPMI. An initial opacity measurement was performed on each of the corneas using an opacitometer (MC2, Clermont, France). Three corneas with opacity readings approximately equivalent to the median opacity of all corneas were selected as negative-control corneas. The opacity of each cornea was read against an air-filled chamber and recorded. Corneas that have an initial opacity reading above 7 units were not dosed. The medium was removed from the anterior chamber and replaced with the test item or control.

750 µL of the test item preparation or the control substance was introduced into the anterior chamber. After 4 hours ± 5 minutes incubation at 32 ± 1°C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed.
After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1°C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.

Test Groups:

- 3 corneas for the test item
- 3 corneas as negative controls treated with physiological saline 0.9% NaCI
- 3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCI
- The BCOP assay is considered to be valid if the in vitro score obtained with the positive control falls within the two standard deviations of the current historical mean.

Vehicle:

physiological saline

Controls:

other: 3 corneas for positive control and negative controls

Amount / concentration applied:

20% solution in saline

Duration of treatment / exposure:

750 µL of the test item preparation or the control substance was introduced into the anterior chamber. After 4 hours ± 5 minutes incubation at 32 ± 1°C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed.

After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1°C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.

Observation period (in vivo):

N/A

Number of animals or in vitro replicates:

3 corneas for Test Item

Details on study design:

See attached report

Irritant / corrosive response data:

Severe irritant

Other effects:

N/A

The eye irritancy potential of FHP-OHS was investigated in the bovine corneal opacity and permeability assay.   The test item was dissolved in physiological saline 0.9% NaCI to gain a 20% concentration.   The following mean in vitro score was calculated: 59.54   Therefore the test item was classified as severe irritant.   Thein vitroscore obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.   For detailed data see Tables 2 - 4 in Appendix 1 of attached report.

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Conclusion: According to the evaluation criteria the test item FHP-OHS is classified as severe eye irritant.

According to GHS (Globally Harmonized Classification System) [3] [9] the test item FHP-OHS is classified into Category 1.

[3] OECD Guideline for the Testing of Chemicals, number 437 “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants” (adopted: 07 September 2009

[9] Globally Harmonized System of Classification and Labelling of Chemicals (GHS), New York & Geneva: United Nations Publications, UN (2007)

Executive summary:

Summary Results: The eye irritancy potential of FHP-OHS was investigated in the bovine corneal opacity and permeability assay. - Preparation of the test item: dissolved in physiological saline 0.9% NaCI - Test item concentration: 20% - Mean in vitro score: 59.54 - Classification: severe irritant - The in vitro score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.   - Conclusion: According to the evaluation criteria the test item FHP-OHS is classified as severe eye irritant.   According to GHS (Globally Harmonized Classification System) [3] [9] the test item FHP-OHS is classified into Category 1.   [3] OECD Guideline for the Testing of Chemicals, number 437 “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants” (adopted: 07 September 2009   [9] Globally Harmonized System of Classification and Labelling of Chemicals (GHS),&: United Nations Publications, UN (2007)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Justification for selection of skin irritation / corrosion endpoint:
Key study

Justification for selection of eye irritation endpoint:
Only study

Effects on eye irritation: highly irritating