Sodium 6,14-diethyl-1,1,1,2,2,18,18,19,19,19-decafluoro-8,12-dioxo-10-({[1-(2,2,3,3,3-pentafluoropropoxy)butan-2-yl]oxy}carbonyl)-4,7,13,16-tetraoxanonadecane-9-sulfonate

Endpoint summary

Description of key information

Eye irritation

in vitro, OECD 437 BCOP, IVIS mean 4.7: not an ocular severe irritant (no corrosive potential)

Skin irritation

in vitro, OECD 439 RHEM, mean viability: 99.07 %: not irritant to skin

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 JAN 2011 - 29 JUL 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Skinethic Skin Irritation Test -42bis from SkinEthic Laboratories (Nice, France).
- Source: adult human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Tissue batch number(s): 11 022A 0209

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 mL PBS, washed once
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/L
- Incubation time: 3 hour
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm

VALIDITY CRITERIA
Negative control (NO acceptance criteria: The NC data meet the acceptance criteria if the mean OD value of the 3 tissues is ≥ 1.2 at 570 nm. The standard deviation value is considered as valid if it is ≤ 18 %, according to the Performance Standards (ECVAM, 2009).
Positive control (PC) acceptance criteria: The PC data meet the acceptance criteria if the mean viability, expressed as % of the NC, is < 40 % and the standard deviation value is ≤ 18 %.

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating or corrosive to skin if the viability after exposure and post-treatment incubation is ≤ 50%.
- The test substance is considered to be non-corrosive and non-irritant to skin if the viability after exposure and post-treatment incubation is > 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 10 µL deionised water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% Sodiumdodecylsulfate-solution in deionised water

Duration of treatment / exposure:

42 min

Duration of post-treatment incubation (if applicable):

42 ± 1 hour

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean 1-3

Value:

99.07

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

MTT-colour assay:

The visual evaluation in the pretest for MTT-reducing capacity of the test material after 3 hours incubation with MTT-reagent did not show blue color.

Validity of the test:

After treatment with the negative control the absorbance values reached an OD of 1.82 (standard deviation 0.06). Therefore, the negative control fulfilled the validity criteria.

Treatment with the positive control induced a sufficient decrease in the relative absorbance up to 1.51 % (standard deviation 0.21), thus the positive control reached the validity criteria.

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

Under the experimental conditions reported, the test material is not irritant to skin.

Executive summary:

Purpose

The in vitro study was performed to assess the irritation potential of the test material by means of the Human Skin Model Test according to OECD Guideline 439 and GLP criteria.

Study Design

The test consisted of a topical exposure of the test item to a human reconstructed model followed by a cell viability test. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential.
Triplicates of the human skin model RHE (Reconstructed Human Epidermis model) were treated either with the test item, the negative or the positive control for 42 minutes. 16 µL of either the negative control (PBS-buffer) or the positive control (5% sodium dodecylsulphat solution) were applied to each tissue. Before adding the test item, 10 µL of deionised water was spread to the epidermis surface to improve further contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to each tissue.

Results

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system.
After treatment with the negative control the absorbance values reached the required acceptability criterion of a mean optical density (OD) ≥ 1.2 for the treatment interval thus showing the quality of the tissues.
The tissue viability after treatment with the test item was higher than 50 % (mean viability: 99.07 %). Therefore, the test item is not considered to possess an irritant potential.

Conclusions

Under the experimental conditions reported, the test item is not irritant to skin.

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:

02 FEB - 16 MAR 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

7 SEP 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

30 MAY 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: slaughterhouse (Odenwaldschlachthof Brensbach, D-64395 Brensbach)
Freshly isolated bovine eyes of donor cattle. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium.

Vehicle:

other: 0.9% sodium chloride solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 20 % in 0.9 % sodium chloride

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 0.9 %
- Lot/batch no. (if required): 0191C15

Duration of treatment / exposure:

incubation for 240 min

Duration of post- treatment incubation (in vitro):

permeability determination: 90 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The corneas were prepared immediately after delivery of the eyes to the laboratory. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (EMEM, pre-warmed at 32 °C).
Each cornea was mounted in a cornea holder (LAB Research, Hungary) with the endothelial side against the sealing ring (0-ring) of the posterior part of the holder. The cornea was gently flattened over the 0-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium (EMEM). The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
Yes, Opacity Measurement before Treatment:
The opacitometer measured the light transmission passing through the corneas and displayed a lux value. This value was recorded in a table and converted into an opacity value (baseline opacity values). The opacitometer was calibrated as described in the manual and the measured values were within the range of the acceptance criteria. The opacity of each cornea was determined by reading each holder placed in the photoreceptor compartment of the opacitometer. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity > 7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated. Three corneas with opacity values close to the median value for all corneas as were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control groups.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
yes, see solvent control

SOLVENT CONTROL USED (if applicable)
yes, 0.9 % sodium chloride solution

POSITIVE CONTROL USED
yes, Imidazole

APPLICATION DOSE AND EXPOSURE TIME
Test material: 20 % suspension in 0.9 % sodium chloride, 240 min
Solvent control: 0.9 % sodium chloride, 240 min
Positive control: 20 % Imidazole solution, 240 min

POST-INCUBATION PERIOD: 90 min (for permeability determination)

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 (or until no visual evidence of test chemical can be observed)
- POST-EXPOSURE INCUBATION: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: baseline opacity was determined with an opacitometer (BASF-OP2.0)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (BioTek ELx800) (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: A substance that induced an IVIS ≥ 55.1 is defined as a corrosive or a severe irritant.

VALIDITY CRITERIA: The test is acceptable if the IVIS of the positive control and the negative control falls within two standard deviations of the current historical mean (IVIS positive control: 90.2 - 148.5; IVIS negative control: -0.9 - 4.2).

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

5.879

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

see vehicle control

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

3.991

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

see vehicle control

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

4.233

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

see vehicle control

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

4.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

see vehicle control

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

4.851

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

see vehicle control

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

3.913

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

see vehicle control

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

3

Value:

1.109

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

see vehicle control

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

1

Value:

0.069

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

see vehicle control

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

2

Value:

0.005

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

see vehicle control

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

3

Value:

0.208

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

see vehicle control

Positive controls validity:

valid

Validity of the Test

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.1 and, thus, within two standard deviations of the current historical mean (IVIS: -0.9 - 4.2). After treatment with the positive control (20% Imidazole) the calculated IVIS was 94. 7 and, thus, also within two standard deviations of the current historical mean (IVIS 90.2 - 148.5). Therefore, the study fulfilled the validity criteria.

Purpose

Thisin vitroGLP study was performed to assess the ocular severe irritant or corrosive potential of the test item by means of the BCOP (Bovine Corneal Opacity and Permeability Assay) according to OECD Guideline 437 and the Commission Regulation (EU) No. 1152/2010.

For this purpose fresh bovine corneas were exposed to a 20% suspension of the test material and toxic effects to the cornea were measured by decreased light transmission (opacity) and increased passage of sodium fluorescein dye (permeability). The opacity and permeability assessments of the corneas were combined to determine anIn VitroIrritancy Score (IVIS), which was used to classify the irritancy potential of the test material.

 

Study Design

In this study the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test material as a 20% suspension in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% imidazole was used. 3 corneas were used per group (test material, negative and positive control group). After a first opacity measurement of the fresh bovine corneas, 750 µL of the suspended test material, positive or negative control were applied on the corneas and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase the test material, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of 1 mL of a fluorescein solution for 90 minutes at 32 ± 1 °C. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS). 

Results

The treatment of the corneas with the negative control (0.9% sodium chloride solution) showed neither an increase of opacity nor an increase of permeability. After treatment with the positive control (20% imidazole) the calculated IVIS was 94.7 and, thus, within two standard deviations of the current historical mean (IVIS: 90.2 - 148.5). Therefore, the study fulfilled the validity criteria. The IVIS obtained after treatment with the test material was 4.7 and, thus, lower than 55.1. Therefore, the test material is not considered to possess an ocular severe irritant or corrosive potential. 

Conclusions

Under the given experimental conditions, the test material did not show an ocular severe irritant or corrosive potential.

Interpretation of results:

other: not Category 1 (irreversible effects on the eye) based on EU GHS criteria

Conclusions:

The IVIS obtained after treatment with the test material was 4.7 and, thus, lower than 55.1. Therefore, the test material is not considered to possess an ocular severe irritant or corrosive potential.

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation - in vitro

Purpose

The in vitro study was performed to assess the irritation potential of the test material by means of the Human Skin Model Test according to OECD Guideline 439 and GLP criteria.

Study Design

The test consisted of a topical exposure of the test item to a human reconstructed model followed by a cell viability test. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential.
Triplicates of the human skin model RHE (Reconstructed Human Epidermis model) were treated either with the test item, the negative or the positive control for 42 minutes. 16 µL of either the negative control (PBS-buffer) or the positive control (5% sodium dodecylsulphat solution) were applied to each tissue. Before adding the test item, 10 µL of deionised water was spread to the epidermis surface to improve further contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to each tissue.

Results

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system.
After treatment with the negative control the absorbance values reached the required acceptability criterion of a mean optical density (OD) is greater than or equal 1.2 for the treatment interval thus showing the quality of the tissues.

The tissue viability after treatment with the test item was higher than 50 % (mean viability: 99.07 %). Therefore, the test item is not considered to possess an irritant potential.

Conclusions

Under the experimental conditions reported, the test item is not irritant to skin.

Eye irritation - in vitro

Purpose

This in vitro GLP study was performed to assess the ocular severe irritant or corrosive potential of the test item by means of the BCOP (Bovine Corneal Opacity and Permeability Assay) according to OECD Guideline 437 and the Commission Regulation (EU) No. 1152/2010.

For this purpose fresh bovine comeas were exposed to a 20% suspension of the test material and toxic effects to the comea were measured by decreased light transmission (opacity) and increased passage of sodium fluorescein dye (permeability). The opacity and permeability assessments of the comeas were combined to determine an In Vitro Irritancy Score (IVIS), which was used to classify the irritancy potential of the test material.

Study Design

In this study the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test material as a 20% suspension in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% imidazole was used.

3 corneas were used per group (test material, negative and positive control group).

After a first opacity measurement of the fresh bovine corneas, 750 µL of the suspended test material, positive or negative control were applied on the corneas and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase the test material, the positive, and the negative control were rinsed from the corneas and the opacity was measured again.

After the opacity measurements, the permeability of the corneas was determined by application of 1 mL of a fluorescein solution for 90 minutes at 32 ± 1 °C. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically.

The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

Results

The treatment of the corneas with the negative control (0.9% sodium chloride solution) showed neither an increase of opacity nor an increase of permeability. After treatment with the positive control (20% imidazole) the calculated IVIS was 94.7 and, thus, within two standard deviations of the current historical mean (IVIS: 90.2 - 148.5). Therefore, the study fulfilled the validity criteria.

The IVIS obtained after treatment with the test material was 4.7 and, thus, lower than 55.1. Therefore, the test material is not considered to possess an ocular severe irritant or corrosive potential.

Conclusions

Under the given experimental conditions, the test material did not show an ocular severe irritant or corrosive potential.

Justification for classification or non-classification

Based on the result of the available in vitro study on skin irritation (RhE-Test Method), the registered substance is not subject to classification as skin irritant in accordance with Regulation (EC) No 1272/2008. According to the result of the available in vitro study on eye irritation (BCOP), a classification as Cat. 1 " Causes serious eye damage" is not warranted. The study allows judgement on severity of effects but not persistence of effects and it does not allow identification of Cat. 2 specifically but since the dertermined value is comparrable to the negatove controls and comparable to the historically negative controls the substance is not expected to have a irritation potential and will not be classified.