2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptan-1-ol

Endpoint summary

Description of key information

irritating to skin - determined in vitro

irritating to eyes - determined in vitro

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

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:

2015

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

commercially available test system

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany). The model used for this study has a functional stratum comeum with an underlying layer of living cells as recommended by the test guidelines. The barrier function of the stratum comeum is adequate, as has been shown by the supplier.
- Tissue batch number(s): Cat.-No.CS-1001
- Date of initiation of testing: 26 March 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: incubator (37 ± 2° C) for 20 min- exposure
- Temperature of post-treatment incubation (if applicable): 37 ± 2° C

PRE-CHECK FOR POTENTIAL OPTICAL INTERFERENCE OF THE TEST ITEM
Optical properties of the test item or its chemical action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability. The test item was therefore tested in advance for a potential direct influence on the test results not related to cytotoxic effects on tissue cells. For this pre-check the following parameters were tested:
1. Assessment of potential direct MTT-reduction of the test item
In case of a direct MTT-reduction of the test item a killed tissue control (inserts, which were killed by freezing) was used in the main assay.
2. Assessment of potential interference of colored or staining test items, which become colored after application to the tissues, with OD read out
In case of an influence of test item color on OD measurement, a color control was used in the main assay.
2.1. Assessment of the color reaction with water
2.2. Assessment of the color reaction with isopropanol

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
the optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.

ASSAY ACCEPTANCE CRITERIA:
The following acceptance criteria determined the validity of an assay:
- mean OD negative control is equal or greater 1.0 and equal or lower 2.8
- mean relative viability of the positive control is equal or lower 20 %
- If the mean viability of the 3 replicates is > 20% the coefficient of variation (CV) should not exceed 0.3.

PREDICTION MODEL / DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- Depending on the controls performed (see 4.4.6) the% Viability Final is calculated as follows (the values always refer to% viability)
% Viability Final = PG - (PG KC - NC KC)
- Thus the test item is identified as irritant and requiring classification according to UN GHS (Category 2 or Category 1) ifthe mean percent tissue viability after exposure and post-treatment incubation is less than or equal to 50 %. In case the test item was found to be non-corrosive (e.g. based on TG 431) and shows tissue viability after exposure and post-treatment incubation which is less than or equal to 50 %, the test item is considered to be irritant to skin in accordance with UN GHS Category 2.

The irritating potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure to the topically applied test item.
All tests were performed in triplets for each time point. The test item was applied for 20 min. Cell viability was measured by the amount of MTT reduction (calculated on the basis of optical density of the negative control).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

other:

Amount/concentration applied:

TEST MATERIAL APPLICATION
- Amount(s) applied: 30 µL of the test item, positive and negative control; a piece of nylon mesh was used as spreading aid

PROCEDURE FOR KILLED CONTROL
The test item was applied to two killed control tissue inserts and treated as described for the main assay (PG KC). In addition two killed control inserts were treated with 0.9 % NaCl (Negative control killed control, NC KC)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μl
- Concentration (if solution): 0.9% NaCl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5 % Sodium Dodecyl Sulfate (SDS)

Duration of treatment / exposure:

20 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

result for test material

Value:

2.13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative controls: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Sample No.	test item	cell viability (%)
1 -3	negative control (0.9% NaCl solution)	100
6 -8	positive control (5% SDS)	1.87
18 -20	test item	2.13

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Executive summary:

An in vitro study for predicting non-specific irritating properties of the test item was conducted according to OECD TG 439. The liquid test item was applied topically to a reconstructed human epidermis model (RhE; epiCS) in an amount of 30µL. The cell viability was determined with 2.13 % for the test item as measured by MTT conversion. It is therefore concluded that according to the criteria of this assay the test item shows irritating properties to the skin and thus requires a classification according to UN GHS (Category 2).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

commercially available test system

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany). The model used for this study has a functional stratum comeum with an underlying layer of living cells as recommended by the test guidelines. The barrier function of the stratum comeum is adequate, as has been shown by the supplier.
- Tissue batch number(s): Cat.-No.CS-1001
- Date of initiation of testing: 22 April 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: incubator (37 ± 2° C) for 60 min- exposure and at room temperature for 3 min- exposure
- Temperature of post-treatment incubation (if applicable): 37 ± 2° C

PRE-CHECK FOR POTENTIAL OPTICAL INTERFERENCE OF THE TEST ITEM
Optical properties of the test item or its chemical action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability. The test item was therefore tested in advance for a potential direct influence on the test results not related to cytotoxic effects on tissue cells. For this pre-check the following parameters were tested:
1. Assessment of potential direct MTT-reduction of the test item
In case of a direct MTT-reduction of the test item a killed tissue control (inserts, which were killed by freezing) was used in the main assay.
2. Assessment of potential interference of colored or staining test items, which become colored after application to the tissues, with OD read out
In case of an influence of test item color on OD measurement, a color control was used in the main assay.
2.1. Assessment of the color reaction with water
2.2. Assessment of the color reaction with isopropanol

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
the optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.

ASSAY ACCEPTANCE CRITERIA:
The following acceptance criteria determined the validity of an assay:
- mean OD negative control is equal or greater 0.8 and equal or lower 2.8
- mean relative viability of the positive control after 60 min is equal or lower 20 %
- If the mean viability of the 3 replicates is eaqual or below 20% the coefficient of variation (CV) should not exceed 0.3.

PREDICTION MODEL / DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- Depending on the controls performed the% Viability Final is calculated as follows (the values always refer to% viability)
% Viability Final = PG - (PG KC - NC KC)
For interpretation of the cell viability results the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test chemicals (or discriminating between different corrosive sub-categories) given in OECD TG 431 were used.

The corrosive potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure to the topically applied test item.
All tests were performed in triplets for each time point. The test item was applied for 3 and 60 min. Cell viability was measured by the amount of MTT reduction (calculated on the basis of optical density of the negative control).

EVALUATION CRITERIA - Step 1
viability < 50% after 3 min exposure - prediction to be considered: corrosive
viability ≥ 50% after 3 min exposure AND < 15% after 60 min exposure - prediction to be considered: corrosive
viability ≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure - prediction to be considered: non-corrosive

EVALUATION CRITERIA - Step 2 (for test items identified as corrosive in Step 1)
viability < 15% after 3 min exposure - prediction to be considered: optional sub-category 1A
viability ≥ 15% after 3 min exposure - prediction to be considered: sub-category 1B (or 1C)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

other:

Amount/concentration applied:

TEST MATERIAL APPLICATION
- Amount(s) applied: 50 µL of the undiluted liquid test item

PROCEDURE FOR KILLED CONTROL
The test item was applied to two killed control tissue inserts and treated as described for the main assay (PG KC). In addition two killed control inserts were treated with 0.9 % NaCl (Negative control killed control, NC KC)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): 0.9% NaCl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution): KOH, 8N

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

treatment time 3 min

Value:

98.66

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

treatment time 60 min

Value:

33.13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative controls: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Sample No.	test item	cell viability (%) after 3 min exposure	cell viability (%) after 60 min exposure
1 -3	negative control (0.9% NaCl solution)	100	100
6 -8	positive control (KOH, 8N)	---	0.35
18 -20	test item	98.66	33.13

Interpretation of results:

GHS criteria not met

Executive summary:

An in vitro study for predicting non-specific corrosive properties of the test item was conducted according to OECD TG 431. The liquid test item was applied topically to a reconstructed human epidermis model (RhE; epiCS) in an amount of 50µL. The cell viability was determined with 98.66% for 3 min exposure and with 33.13% for 60 min exposure to the test item as measured by MTT conversion. It is therefore concluded that according to the criteria of this assay the test item shows no corrosive properties to the skin and thus requires no classification according to UN GHS.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: three-dimensional human cornea model tissue model

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability:
In principle the EpiOcular™ eye irritation test (EIT) measures the ocular irritation potential of a test item by determination of cytotoxic effects on a reconstructed human cornea epithelium (RhCE) tissue model to discriminate chemicals not requiring classification for eye irritancy (UN GHS No Category) from those requiring classification. The EpiOcular™ EIT is not intended to differentiate between UN GHS Category 1 (serious eye damage) and UN GHS Category 2 (eye irritation). This differentiation would need to be addressed by another tier of a test strategy. As a BCOP has already been conducted and Category 1 can be excluded based on the result, this test can be used to determine, if the substance needs to be classified (Category 2) or not.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The experiment was carried out on the EpiOcular™ RhCE tissue construct (about 0.6 cm² in size; MatTek Corporation, Slovakia).
The RhCE tissue equivalents were shipped in 24 well cell culture plates on semi solid agar’s medium (Lot No.: 30660).

Experimental starting date: 26 May 2020

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: no Killed Control (KC), Colour Control (CC) and Non-Specific Killed Control (NS KC) needed

Amount / concentration applied:

50 µL per insert

Duration of treatment / exposure:

30 min at standard culture conditions (5 % CO2, 37 °C, 95 % humidity) followed by a post-soak immersion period of about 12 min in fresh medium

Duration of post- treatment incubation (in vitro):

120 min (37 °C, 5 % CO2, 95 % humidity)

Number of animals or in vitro replicates:

each 2 tissues for the test item, positive and negative controls

Details on study design:

- RhCE tissue construct used, including batch number: The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier.
RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.

- Doses of test chemical and control substances used: 50 µg of the neat test item, 50 µL negative control (deionized water) and positive control (neat methyl acetate), respectively, were applied to the EpiOcular™ tissue surface in duplicate.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): 37 ± 2 °C; 6 h exposure, 25 min. post-soak immersion, 18 h post-treatment incubation

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): Killed control and colour control not required.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): each 2 tissues for the test item, positive and negative controls

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer (EL808, Bio-Tek; 96 well format, 200 µL).

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: For interpretation of cell viability results the cut-off value distinguishing classified (irritant) from non-classified substances as given in OECD TG 492 was used:
- The test chemical is identified as not irritant and not requiring classification according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60 %.
- The test chemical is identified as irritant and potentially requiring classification according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60 %.

- Positive and negative control means and acceptance ranges based on historical data: The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.5
- mean relative viability of the positive control (PC) is < 50 % (relative to negative control)
- the difference of viability between the two replicates is < 20 %.

Irritation parameter:

other: cell viability (%)

Run / experiment:

mean

Value:

33.74

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 % viability

Positive controls validity:

valid

Remarks:

14.13 % viability

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The study was conducted under GLP according to OECD guideline 439 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritation potential of the test item to the eye in vitro.
As the final test item-treated tissue viability was 33.74 % relative to negative control, the test item can be characterized as having eye irritating properties.

Executive summary:

The model used is standardized and commercially available. The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD 492. The liquid test item was applied topically to the RhCE tissue surface in duplicate for 30 min, followed by an 120 min post-treatment incubation period. Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which was set at 100 %.

The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) of the tissue model. The final mean percent tissue viability recorded for the test item is 34 % (rounded).

According to the results of this study the test item was identified as requiring classification for eye irritation according to UN GHS (Category 2 or 1).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2017

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl per cornea (anterior chamber of the cornea holder) for liquid test item, negative control and positive control

Immediately before application, the medium was aspirated from the anterior chamber. 750 μL of negative and positive control and of the test item were applied to the corneas through the holes of the anterior chamber. The holes of both chambers were sealed with adhesive tape and the holders were kept with the front side up, so that the liquids covered the cornea sufficiently. The holders were transfe1Ted into the incubator at 32 °C (± 1 °C) for the exposure
time of 10 minutes.
After the exposure, the negative and positive control and the test item were aspirated from the anterior chamber and the corneas were rinsed at least 3 times with phenol red containing MEM to show effectiveness of test substance removal. During the final rinse cycle the corneas were rinsed again with clear MEM medium in order to remove residues of the dye.

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

3 corneas per tested material

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Bovine eyes of slaughtered cattle were extracted and transferred in containers with Hank’s balanced salt solution (HBSS) with penicillin/streptomycin solution. For transportation the containers were ice cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HBSS supplemented with penicillin/streptomycin solution and 1 % FBS and stored overnight at 2-8 °C. On the next day (day of testing) the containers with the eyes were placed in an incubator at 32 ° C (± 1 ° C) for about 2 hours before preparation of the corneas.
For the preparation of the cornea the sclera was incised with a scalpel and cut by scissors. A 2-3 mm wide scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders were placed for at least 1 hour in the incubator at 32 °C (± 1 °C).

QUALITY CHECK OF THE ISOLATED CORNEAS
yes

NEGATIVE CONTROL USED: physiological saline

POSITIVE CONTROL USED: NaOH (1% in isotonic saline solution v/v)

APPLICATION DOSE AND EXPOSURE TIME
the test materials were applied pure for 4 hours

TREATMENT METHOD:
closed chamber method

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: corneas were rinsed at least 3 times

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer (BASF OP3.0). Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- Corneal permeability: The medium in anterior chamber of each holder was replaced by 1ml of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 °C (± 1 °C) for about 90 minutes. After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 μL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluoresceinsolution was prepared and also filled into the 96-well plate, in triplicates. The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA - Reader).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Test item formulations that cause an IVIS value > 55 are classified as seriously damaging the eye (UN GHS Cat 1). For test item formulations that cause IVIS values of > 3 and ≤ 55 no prediction can be made, indicated by 'No Category 1'. Test item formulations that cause IVIS values of ≤ 3 can be considered according to Guideline as having no irritating potential to the eye, indicated by 'No Category'.

Irritation parameter:

in vitro irritation score

Remarks:

(IVIS)

Run / experiment:

mean of 3 corneas

Value:

13.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

IVIS of 3.2

Positive controls validity:

valid

Remarks:

IVIS of 294.6

Remarks on result:

other: no indication of severe eye damage

tested materials  opacity values per cornea  permeability values per cornea  IVIS per cornea  IVIS mean  negative control (3 corneas)  4.8, 3.2, 1.3  0.007, 0.007, 0.010  4.9, 3.3, 1.4  3.2 (SD 1.7)  positive control (3 corneas) 293.7, 250.1, 263.2  1.256, 2.137, 1.728  312.6, 282.2, 289.1 294.6 (SD 15.9)  test item  13.0, 10.9, 15.2 0.061, 0.033, 0.034  13.9, 11.4, 15.7  13.7 (SD 2.1)

Interpretation of results:

other: negative result: no severe eye damage

Executive summary:

The substance was tested in the BCOP test according to OECD TG 437. For determination of corneal damage tissue opacity as well as tissue permeability of 3 bovine corneas was measured after a 4 hour incubation time with the test item. Based on these data, in comparison to the negative control, the In Vitro Irritancy Score (IVIS) value was calculated with 13.7 which is below the IVIS cut-off threshold of 55 and thus identifying the test item as not inducing serious eye damage.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro studies for predicting non-specific irritating and corrosive properties to skin were conducted according to OECD TG 439 and OECD TG 431. The liquid test item was applied topically to a reconstructed human epidermis model (RhE; epiCS). In the in vitro skin corrosion test the cell viability was determined with 98.66% for 3 min exposure and with 33.13% for 60 min exposure to the test item, resulting in no classification for skin corrosion. The in vitro skin irritation test, however, showed a cell viability of only 2.13 % for the test item. It is therefore concluded that according to the criteria of the assays the substance led to irritating properties and thus requires a classification as skin irritant (Skin Irrit. Cat 2).

The substance was tested ex vivo in the BCOP test according to OECD TG 437. For determination of corneal damage tissue opacity as well as tissue permeability of 3 bovine corneas were measured after a 4 hour incubation time with the test item. Based on these data, in comparison to the negative control, the In Vitro Irritancy Score (IVIS) value was calculated with 13.7 which is below the IVIS cut-off threshold of 55 and thus identifying the test item as not inducing serious eye damage.

The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD TG 492. The liquid test item was applied topically to the RhCE tissue surface in duplicate for 30 min, followed by an 120 min post-treatment incubation period. Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which was set at 100 %.

The final mean percent tissue viability recorded for the test item was 34 %, and thus, taking into accout the result of the BCOP, the substance was identified as requiring classification for eye irritation (Eye Irrit Cat 2).

Justification for classification or non-classification

Based on in vitro/ex vivo skin and eye irritation tests (OECD TG 431, 439, 437 and 492) and according to EU Regulation 1272/2008 the substance has to be classified as skin irritant and eye irritant with Skin Irrit 2 and Eye Irrit 2.