2-Propenal, 2-fluoro-3-(4-morpholinyl)-, (Z)-

Endpoint summary

Description of key information

Skin corrosion/irritation

A study for predicting a non-specific, corrosive potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 431. For the determination of time related cytotoxic effects the incubation periods were 3 min. and 60 min. The MTT (Methylthiazoletetrazolium) viability test results (3 min.: 101.61 % viability; 60 min.: 92.11 % viability) showed, that the test item has no corrosive property under the conditions of the assay used.

A study for predicting a skin irritation potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 439. After an exposure period of 20 minutes, followed by a 42 hours post-treatment incubation period, the mean value of cell viability was measured to be 84.24 % in the MTT (Methylthiazoletetrazolium) conversion assay. Thus, the test item is considered to have no skin irritation category as defined in the UN GHS.

Eye damage/irritation

The test item (20 % (w/v) in isotonic saline) was tested in the BCOP test according to OECD TG 437. For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour exposure time. Based on these data, in comparison to the vehicle control, the mean In Vitro Irritancy Score (IVIS) value was calculated to be 42.6 which is below the IVIS cut-off threshold of 55 and thus identifying the test item as not inducing serious eye damage. The results of the positive (20 % imidazole solution) and negative (isotonic saline solution) controls confirmed the validity of the test system.

An in vitro study for assessing ocular irritation properties of the test item was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcularTM. The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD 492. The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period. According to the conducted pre-check the test item was demonstrated to exert optical interferences directly affecting the test results that are not related to cytotoxic effects on tissue cells. Therefore a killed control,a color control and a non-specific killed control had to be conducted. 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 viability value for the test item was then calculated by subtracting the appropriate controls from the test item treated values (color control (CC) I killed control (KC)) and added the Non-specific killed control NS KC as follows: Final viability test item = % viability test item - (%viability test item KC - % viability NCKC) - % viability test item CC + viability NS KC. 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. As the final mean percent tissue viability recorded for the test item is below 60% (2.5%) relative to the negative control, the test item was characterized as having eye irritating properties.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

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 method

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Cat.-No: CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

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

PREDICTION MODEL / DECISION CRITERIA:
- Corrosivity potential of test materials is predicted from the cell viabilities obtained after 3 min and 60 min treatment compared to the negative control. A chemical is classified "corrosive" (sub-category 1A) if the cell viability after 3 min treatment is decreased by more than 50 %. If cell viability after 3 min exposure is ≥ 50 %, while it is below 15 % after 60 min exposure the substance is also classified as corrosive, but sub-category 1 B/1 C. If cell viability after 3 min exposure is ≥ 50 % and after 60 min exposure ≥ 15 %, the substance is classified as non-corrosive.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Reliability of the test was previously confirmed by interlaboratory validation

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg (plus 30 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)

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): 8N KOH

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

cell viability after 3 min [%]

Value:

ca. 102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

cell viability after 60 min [%]

Value:

ca. 92

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction and Colour interference with MTT: Since the test substance is a dark-colored test item the preliminary tests cannot be performed. Therefore in the main assay all controls were carried out.

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

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

Table 1: Tabular summary of results   60 min

Sample No.	Test item	OD mean	Std Dev	% Viability
1 - 3	Negative control NaCl 0.9 %	1.89	0.01	100.00
4 - 5	NC KC NaCl 0.9%	0.03	0.00	1.49
6 - 8	PC 8N KOH	0.01	0.00	0.58
9 - 11	Fluormorpholinacrylaldehyd	1.74	0.03	92.06
12 - 13	Fluormorpholinacrylaldehyd KC	0.03	0.00	1.44
14 -15	Fluormorpholinacrylaldehyd CC	0.01	0.00	0.29
12 - 13	Fluormorpholinacrylaldehyd NsKC	0.01	0.00	0.29

Table 2: Tabular summary of results  3 min

Sample No.	Test item	OD mean	Std Dev	% Viability
18- 20	Negative control NaCl 0.9 %	2.01	0.03	100.00
21- 22	NC KC NaCl 0.9%	0.03	0.00	1.57
23 - 25	Fluormorpholinacrylaldehyd	2.04	0.08	101.55
26 - 27	Fluormorpholinacrylaldehyd KC	0.03	0.00	1.61
28 - 29	Fluormorpholinacrylaldehyd CC	0.01	0.00	0.38
30 -31	Fluormorpholinacrylaldehyd NsKC	0.01	0.00	0.49

Interpretation of results:

other: negative

Executive summary:

A study for predicting a non-specific, corrosive potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 431. For the determination of time related cytotoxic effects the incubation periods were 3 min. and 60 min. The MTT (Methylthiazoletetrazolium) viability test results (3 min.: 101.61 % viability; 60 min.: 92.11 % viability) showed, that the test item has no corrosive property under the conditions of the assay used.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2018

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)

Deviations:

no

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 method

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany)
- Cat.-No: CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

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 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.

PREDICTION MODEL / DECISION CRITERIA
- The mean optical density (OD) values obtained with the test item were used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- According to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg (plus 30 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5% SDS in physiological saline

Duration of treatment / exposure:

20 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

cell viability after 20 min [%]

Value:

ca. 84

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No - according to the results of the pre-check
- Colour interference with MTT: No - according to the results of the pre-check

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

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

Table 1: Tabular summary of the results    

Sample No.	Test item	OD mean	Std Dev	% Viability
1 - 3	Negative control NaCl 0.9 %	1.75	0.02	100.00
4 - 5	NC KC NaCl 0.9%	0.03	0.00	1.65
6 - 8	Positive control SDS 5 %	0.06	0.02	3.59
9 - 11	Fluormorpholinylacrylaldehyde	1.47	0.02	83.68
12 - 13	Fluormorpholinylacrylaldehyde KC	0.02	0.00	1.10
14 - 15	FluormorpholinylacrylaldehydebCC	0.01	0.00	0.37
16 - 17	Fluormorpholinylacrylaldehyde NsKC	0.01	0.00	0.37

Interpretation of results:

other: negative

Executive summary:

A study for predicting a skin irritation potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 439. After an exposure period of 20 minutes, followed by a 42 hours post-treatment incubation period, the mean value of cell viability was measured to be 84.24 % in the MTT (Methylthiazoletetrazolium) conversion assay. Thus, the test item is considered to have no skin irritation category as defined in the UN GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

weight of evidence

Study period:

August 2018

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: Analytical determination of stability and homogeneity of the test item in the vehicle was not performed specifically for this study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was suspended with a whirl mixer prior to application in isotonic saline solution to achieve a concentration of 20 % (w/v). During application the suspension was stirred on a magnet stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): The preparation was visually described as suspension.

Details on test animals or tissues and environmental conditions:

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.

Vehicle:

physiological saline

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 and chamber
- Concentration (if solution): 20 % (w/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 μL per cornea and chamber
- Concentration (if solution): 0.9% NaCl (w/v)

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

no further incubation required

Number of animals or in vitro replicates:

3 cornea

Details on study design:

Immediately before application, the medium was aspirated from the anterior chamber. 750 μL of the test material formulations were applied to the corneas, each 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 formulations covered the cornea sufficiently (closed chamber method). The holders were transferred into the incubator at 32 °C (± 1 °C) for the exposure time of 4 hours. After the exposure, the formulations 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 pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. Before measuring opacity, fresh MEM medium was filled in the chambers.

Positive control: 20 % Imidazole in physiologic saline (w/v; 750 µL)

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

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 (with integrated light meter testo 545 and Comfort 3.4 SP6 software from Testo AG, Lenzkirch). 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 (Bio-Tek EL 808, Software Gen5).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:decision criteria as indicated in the OECD TG 437

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

42.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: A yellow coloring of the corneas, treated with 20% test substance, was observed.

Table 1: Individual values of opacity, permeability and IVIS

Cornea No.	Opacity per cornea	Permeability per cornea	IVIS per cornea	IVIS per group
				mean		SD
Negative control                    1	-2.8	0.016	-3.9
0.9 % NaCl                           2	0.6	0.007	1.2	-1.0		1.6
3	-1.2	0.008	-2.4
Positive Control                     4	112.0	1.104	93.3
20 % Imidazole                      5	103.7	0.797	89.0	123.25		6.9
6	100.1	1.738	104.0
Test item                                 7	32.9	0.879	-0.3
20%   Fluormorpholinyl- 8 acrylaldehyde	21.3	0.367	5.1	42.6		14.4
9	28.0	1.800	4.4

 

Interpretation of results:

other: negative

Executive summary:

The test item (20 % (w/v) in isotonic saline) was tested in the BCOP test according to OECD TG 437. For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour exposure time. Based on these data, in comparison to the vehicle control, the mean In Vitro Irritancy Score (IVIS) value was calculated to be 42.6 which is below the IVIS cut-off threshold of 55 and thus identifying the test item as not inducing serious eye damage. The results of the positive (20 % imidazole solution) and negative (isotonic saline solution) controls confirmed the validity of the test system.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

September 2018

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)

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: reconstructed human cornea-like epithelium (RhCE)

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

JUSTIFICATION OF THE TEST METHOD AND CONSIDERATIONS REGARDING APPLICABILITY :
The EpiOcular™ eye irritation test (EIT) follows international OECD test guidelines. The 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).

DESCRIPTION OF THE CELL SYSTEM USED, INCL. CERTIFICATE OF AUTHENTICITY AND THE MYCOPLASMA STATUS OF THE CELL LINE :
The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The model is standardized and commercially available. 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 (MatTek Corporation, Slovakia).

ENVIRONMENTAL CONDITIONS :
The environmental conditions in the incubator were standardized as follows:
Incubator temperature: 37 +/- 2° C
CO2 gas concentration: 5 %
Humidity: maximum
All incubation steps were performed in a CO2 atmosphere incubator (Heraeus, Osterode, Germany).

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): 50 mg per tissue insert in duplicate
- Concentration (if solution): neat test item

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 μLper tissue insert in duplicate

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

tissue inserts were used in duplicate for test item, negative and positive control

Details on study design:

RhCE TISSUE CONSTRUCT USED, INCLUDING BATCH NUMBER :
EpiOcular RhCE tissue supplied by MatTek Corporation,

PRE-CHECK FOR POTENTIAL OPTICAL INTERFERENCES 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
2.1. Assessment of the color reaction with water
2.2. Assessment of the color reaction with isopropanol
In case of an influence of test item color on OD measurement, a color control was used in the main assay.
The evaluation criteria for the pre-check were:
- For MTT reduction (visual assessment): If the MTT solution color turns blue/purple, the test item is presumed to have reduced the MTT. A killed control must be conducted.
- For color reaction (measurement of the OD): If, after subtraction of the OD for water or isopropanol the OD of the test item solution is >0.08 a Color Control must be conducted.

If a test item is identified as producing both, color interference and direct MTT reduction, a third set of controls (Non-specific killed control NS KC) is required, additionally from the PG KC and PG CC controls. This is usually the case with darkly coloured test items interfering with the MTT assay (e.g. blue, purple, black) because their intrinsic colour impedes the assessment of their capacity to directly reduce MTT.

PROCEDURE FOR KILLED CONTROL OR COLOR CONTROL :
Color control (CC): The test item was applied to two additional EpiOcular™ tissue inserts which were in principle treated as described for the main assay. However the further processing for the determination of cell viability differs in that these color control inserts were placed in Assay medium instead of placing them in MTT solution.
Killed control (KC): The test item was applied to two killed control tissue inserts and treated as described for the main assay. In addition two killed control inserts were treated with sterile deionized water ('Negative control killed control, NC KC').
Non specific killed control (NS KC): The test item was applied to two killed epiCS inserts which were in principle treated as described for the main assay. However the fmther processing for the determination of cell viability differs in that these control inserts were placed in MTT-Assay medium instead of placing them in MTT solution.

DESCRIPTION OF THE METHOD USED TO QUANTIFY MTT FORMAZAN :
For viability testing the inserts were placed in new plates containing MTT solution (1 mg/ml in Maintenance medium at 37°C). The tissues were incubated for 180 ± 10 min. under standard culture conditions. The extraction of blue formazan was performed in isopropanol on a vertical shaker for 2-3 hours at room temperature. 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). Data acquisition and evaluation were performed with the software "Gen5" (Bio-Tek).
The OD values obtained with the replicate tissue extracts for the test item were used to calculate the mean percent tissue viability normalized to negative control, which was set at 100%.
The final viability value for the test item was then calculated by subtracting the appropriate controls from the test item treated values (color control (CC) I killed control (KC)) and added the Non-specific killed control NS KC (see section 4.4.5) as follows:
Final viability test item = % viability test item - (%viability test item KC - % viability NC KC) - % viability test item CC + viability NS KC.

ASSAY ACCEPTANCE CRITERIA :
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: final cell viability (%)

Value:

ca. 2

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:

- OTHER EFFECTS:
- Visible damage on test system: No
According to the conducted pre-check the test item was demonstrated to exert optical interferences directly affecting the test results that are not related to cytotoxic effects on tissue cells. Therefore a killed control,a color control and a non-specific killed control had to be conducted.

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

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

Interpretation of results:

other: positive

Executive summary:

An in vitro study for assessing ocular irritation properties of the test item was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcularTM. The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD 492. The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period. According to the conducted pre-check the test item was demonstrated to exert optical interferences directly affecting the test results that are not related to cytotoxic effects on tissue cells. Therefore a killed control,a color control and a non-specific killed control had to be conducted.

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 viability value for the test item was then calculated by subtracting the appropriate controls from the test item treated values (color control (CC) I killed control (KC)) and added the Non-specific killed control NS KC as follows: Final viability test item = % viability test item - (%viability test item KC - % viability NCKC) - % viability test item CC + viability NS KC.

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. As the final mean percent tissue viability recorded for the test item is below 60% (2.5%) relative to the negative control, the test item was characterized as having eye irritating properties.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the study results for skin irritation a classification according to Regulation (EC) No. 1272/2008 (CLP), ANNEX I, is not required.

Based on the study results for eye irritation a classification in Category 2 (irritating to eyes) according to Regulation (EC) No. 1272/2008 (CLP), ANNEX I, is warranted.