Registration Dossier

Administrative data

Description of key information

A GLP-compliant in vitro skin irritation and corrosion turnkey testing strategy was conducted in the EpiDerm Test according to OECD Guideline 431 and OECD Guideline 439 to assess the skin irritating / corrosion potential of the test substance. Based on the results observed in the studies the test substance shows no skin irritation potential in the EpiDerm in vitro skin irritation and corrosion test strategy.

A GLP-compliant in vitro eye irritation and corrosion turnkey testing strategy was conducted according to OECD Guideline 437 (BCOP) and OECD Guideline 492 (EpiOcular) to assess the eye irritation potential of the test substance. Based on the results observed in the studies the test substance shows eye irritating potential in the in vitro eye irritation test strategy.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 0017319511
- Expiration date of the batch: 18 Dec 2018
- Purity: > 99 %
- pH value: ca. 6
- Physical state / color: Solid, wxy / white to yellowish

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: To improve application, the solid waxy test substance was heated at about 50 °C for about 15 minutes. Before application, the test substance was cooled down to room temperature.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The Skin Corrosion Test is based on the experience that corrosive and irritant chemicals produce cytotoxicity in human reconstructed epidermis after a short term topical exposure. The test is designed to predict a skin corrosion or irritation potential of a chemical by using the three-dimensional human epidermis model EpiDerm. After application of the test material to the stratum corneum surface of the EpiDerm tissue, the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow-colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue-colored formazan. After isopropanol extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. The optical density of the extracts of tissues treated with the test substance is compared to negative control values from tissues and expressed as relative tissue viability.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number(s): 25882

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. After incubation, the tissues were washed with PBS to stop the MTT incubation.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.9 mL MTT solution
- Incubation time: 3 hours
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm
- Filter: filter wavelength 570 nm without reference filter

NUMBER OF REPLICATE TISSUES: two tissues per exposure time

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze-killed

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
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): 50 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
Duration of treatment / exposure:
3 min and 1 h
Number of replicates:
2 tissues per exposure time
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure period
Value:
104.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 h exposure period
Value:
59.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: Exposure period 3 min: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance

Identification

 

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Viable tissues

Mean OD570

1.789

1.779

1.784

 

 

Viability

[% of NC]

100.3

99.7

100.0

0.4

0.4

KC tissues

Mean OD570

0.085

0.083

0.084

 

 

Viability

[% of NC]

4.8

4.7

4.7

0.1

1.7

Test substance

Viable tissues

Mean OD570

1.833

1.930

1.882

 

 

Viability

[% of NC]

102.7

108.2

105.5

3.9

3.7

KC tissues

Mean OD570

KC NC corrected

0.020

0.005

0.012

 

 

Viability

[% of NC]

1.1

0.3

0.7

0.6

83.7

Final relative mean viability of tissues after KC correction [% of NC]

104.8

 

 

PC

Viable tissues

Mean OD570

0.179

0.185

0.182

 

 

Viability

[% of NC]

10.0

10.4

10.2

0.2

2.3

Table 2: Exposure period 1 h: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance

Identification

 

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Viable tissues

Mean OD570

1.479

1.668

1.574

 

 

Viability

[% of NC]

94.0

106.0

100.0

8.5

8.5

KC tissues

Mean OD570

0.092

0.069

0.080

 

 

Viability

[% of NC]

5.8

4.4

5.1

1.0

20.3

Test substance

Viable tissues

Mean OD570

1.160

0.856

1.008

 

 

Viability

[% of NC]

73.7

54.4

64.1

13.7

21.3

KC tissues

Mean OD570

KC NC corrected

0.062

0.089

0.075

 

 

Viability

[% of NC]

3.9

5.6

4.8

1.2

24.9

Final relative mean viability of tissues after KC correction [% of NC]

59.3

 

 

PC

Viable tissues

Mean OD570

0.065

0.089

0.077

 

 

Viability

[% of NC]

4.1

5.7

4.9

1.1

22.5

Interpretation of results:
other: No skin corrosion potential observed.
Conclusions:
No prediction can be made for skin corrosion according to GHS criteria based on the results of this in vitro study alone.
Executive summary:

The potential of the test substance to cause dermal corrosion was assessed by a single topical application of 50 µL undiluted test substance to a reconstructed three-dimensional, human epidermis model (EpiDerm).

The test substance could not be homogeneously distributed on the whole application area. Therefore, a metal pin covered with 50 µL undiluted, at ca. 50 °C heated test substance was applied covering the whole tissue surface. Before application, the test substance was cooled down to room temperature.

Two EpiDerm tissues were incubated with the test substance for 3 minutes and 1 hour each. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced. The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 104.8 %, and it was 59.3 % after an exposure period of 1 hour.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Feb - Jul 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 0017319511
- Expiration date of the batch: 18 Dec 2018
- Purity: > 99 %
- pH value: ca. 6
- Physical state / color: Solid, waxy / white to yellowish

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: To improve application, the solid waxy test substance was heated at about 50 °C for about 15 minutes. Before application, the test substance was cooled down to room temperature.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The test is based on the experience that corrosive and irritant chemicals produce cytotoxicity in human reconstructed epidermis after a short term topical exposure. The test is designed to predict a skin corroion or irritation potential of a chemical by using the three-dimensional human epidermis model EpiDerm. After application of the test material to the stratum corneum surface of the EpiDerm tissue, the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow-colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue-colored formazan. After isopropanol extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. The optical density of the extracts of tissues treated with the test substance is compared to negative control values from tissues and expressed as relative tissue viability.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number(s): 25887, 28600, 28623, 28637

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for 25 min and 37 °C for 35 min
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. After incubation with MTT, the tissues were washed with PBS to stop the MTT incubation.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.9 mL MTT solution
- Incubation time: 3 hours
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm
- Filter: Measurement using a filter wavelength 570 nm without reference filter

NUMBER OF REPLICATE TISSUES: three tissues were treated in each test run with the test substance, the PC and the NC, respectively

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): killed by freezing at - 20 °C

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test chemical is identified as requiring classification and labelling 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 %.
- The test substance is considered to be not irritating 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): 30 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 tissues per test run
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st test run
Value:
54.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2nd test run
Value:
52
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3rd test run
Value:
41.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
4th test run
Value:
66.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: 1st test run: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance identification

 

 

Tissue 1

Tissue 2

Tissue 3

Mean

SD

CV [%]

NC

Viable tissues

Mean OD570

1.926

1.720

1.589

1.745

 

 

Viability

[% of NC]

110.4

98.6

91.1

100.0

9.7

9.7

KC tissues

Mean OD570

0.045

0.043

0.044

0.044

 

 

Viability

[% of NC]

2.6

2.5

2.5

2.5

0.0

1.7

Test substance

Viable tissues

Mean OD570

1.110

0.509

1.345

0.988

 

 

Viability

[% of NC]

63.6

29.2

77.1

56.6

24.7

43.6

KC tissues

Mean OD570

KC NC corrected

0.022

0.074

0.023

0.040

 

 

Viability

[% of NC]

1.3

4.3

1.3

2.3

1.7

74.3

Final relative mean viability of tissues after KC correction [% of NC]

54.3

 

 

PC

Viable tissues

Mean OD570

0.048

0.047

0.051

0.049

 

 

Viability

[% of NC]

2.8

2.7

2.9

2.8

0.1

4.3

SD: Standard deviation

CV: coefficient of variation

NC: Negative control

PC: Positive control

Table 2: 2nd test run: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance identification

 

 

Tissue 1

Tissue 2

Tissue 3

Mean

SD

CV [%]

NC

Viable tissues

Mean OD570

1.840

1.460

1.722

1.674

 

 

Viability

[% of NC]

109.9

87.2

102.9

100.0

11.6

11.6

KC tissues

Mean OD570

0.040

0.046

0.045

0.044

 

 

Viability

[% of NC]

2.4

2.7

2.7

2.6

0.2

7.0

Test substance

Viable tissues

Mean OD570

1.123

0.504

1.098

0.908

 

 

Viability

[% of NC]

67.1

30.1

65.6

54.3

21.0

38.6

KC tissues

Mean OD570

KC NC corrected

0.025

0.036

0.053

0.038

 

 

Viability

[% of NC]

1.5

2.1

3.1

2.3

0.8

36.8

Final relative mean viability of tissues after KC correction [% of NC]

52.0

 

 

PC

Viable tissues

Mean OD570

0.051

0.046

0.052

0.049

 

 

Viability

[% of NC]

3.0

2.7

3.1

2.9

0.2

6.9

SD: Standard deviation

CV: coefficient of variation

NC: Negative control

PC: Positive control

Table 3: 3rd test run: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance identification

 

 

Tissue 1

Tissue 2

Tissue 3

Mean

SD

CV [%]

NC

Viable tissues

Mean OD570

1.658

2.106

1.999

1.921

 

 

Viability

[% of NC]

86.3

109.6

104.0

100.0

12.2

12.2

KC tissues

Mean OD570

0.050

0.050

0.049

0.049

 

 

Viability

[% of NC]

2.6

2.6

2.6

2.6

0.0

0.6

Test substance

Viable tissues

Mean OD570

1.162

0.482

0.878

0.840

 

 

Viability

[% of NC]

60.5

25.1

45.7

43.7

17.8

40.6

KC tissues

Mean OD570

KC NC corrected

0.033

0.071

0.020

0.041

 

 

Viability

[% of NC]

1.7

3.7

1.0

2.2

1.4

64.6

Final relative mean viability of tissues after KC correction [% of NC]

41.6

 

 

PC

Viable tissues

Mean OD570

0.049

0.055

0.047

0.050

 

 

Viability

[% of NC]

2.6

2.8

2.4

2.6

0.2

8.2

SD: Standard deviation

CV: coefficient of variation

NC: Negative control

PC: Positive control

Table 4: 4th test run: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance identification

 

 

Tissue 1

Tissue 2

Tissue 3

Mean

SD

CV [%]

NC

Viable tissues

Mean OD570

1.866

2.181

1.617

1.888

 

 

Viability

[% of NC]

98.8

115.5

85.6

100.0

15.0

15.0

KC tissues

Mean OD570

0.053

0.051

0.051

0.052

 

 

Viability

[% of NC]

2.8

2.7

2.7

2.7

0.1

2.8

Test substance

Viable tissues

Mean OD570

1.312

1.439

1.299

1.350

 

 

Viability

[% of NC]

69.5

76.2

68.8

71.5

4.1

5.7

KC tissues

Mean OD570

KC NC corrected

0.091

0.077

0.105

0.091

 

 

Viability

[% of NC]

4.8

4.1

5.5

4.8

0.7

15.1

Final relative mean viability of tissues after KC correction [% of NC]

66.7

 

 

PC

Viable tissues

Mean OD570

0.050

0.048

0.053

0.050

 

 

Viability

[% of NC]

2.6

2.6

2.8

2.7

0.1

5.1

SD: Standard deviation

CV: coefficient of variation

NC: Negative control

PC: Positive control

Interpretation of results:
GHS criteria not met
Remarks:
no indication of skin irritation
Conclusions:
No prediction can be made for skin irritation according to GHS criteria based on the results of this in vitro study alone.
Four test runs of the skin irritation test (SIT) were performed, due to high variability of results.
The non-concordant results within test runs 1 to 3 of the EpiDerm skin irritation test may be attributed to technical issues at removal of the waxy test substance. Wetting the tissues prior to application of the 4th test run led to concordant and unambiguous results.
Overall, the viability values in most tissues (especially all tissues in test run 4) are well above the cut off for skin irritation. Thus, it is concluded that the test substance does not indicate an irritation potential.
Executive summary:

The potential of the test substance to cause dermal irritation was assessed by a single topical application of 30 µL undiluted test substance to a reconstructed three-dimensional, human epidermis model (EpiDerm).

The test substance could not be homogeneously distributed on the whole application area. Therefore, a metal pin covered with 30 µL undiluted, at ca. 50 °C heated test substance was applied covering the whole tissue surface. Before application, the test substance was cooled down to room temperature. Due to technical issues at removal of the waxy test substance during several test runs of the skin irritation test, the tissues were wetted with 25 µL sterile PBS before application of the test substance in a 4th test run.

The irritation test was performed with three EpiDerm tissues per test run, which were incubated with the test substance for 1 hour followed by a 42 -hour post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure / post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced. Four test runs were performed, due to high variability of results.

1st test run: Due to the non-concordant replicate measurements (relative tissue viability) of the test substance treated tissues (values for single tissues (without KC correction): 63.6 %, 29.2 % and 77.1 %), another test run of the EpiDerm skin irritation test was performed to clarify the result.

2nd test run: Again non-concordant replicate measurements (relative tissue viability) of the test substance treated tissues (values for single tissues (without KC correction): 67.1 %, 30.1 % and 65.6 %) were obtained. The 2nd tissue was partly detached during the washing procedure. Thus, another test run of the EpiDerm skin irritation test was performed.

3rd test run: The replicate measurements (relative tissue viability) of the test substance treated tissues were again not concordant (values for single tissues (without KC correction): 60.5 %, 25.1 % and 45.7 %).

The non-concordant results of test run 1 to 3 of the EpiDerm skin irritation test may be attributed to technical issues at removal of the waxy test substance. Thus, the tissues were wetted with 25 µL sterile PBS before application of the test substance in a 4th test run.

4th test run: The final relative mean viability of the tissues treated with the test substance of the 4th test run was 66.7 % (values for single tissues (without KC correction): 69.5 %, 76.2 % and 68.8 %). All acceptance criteria were met. Wetting the tissues prior to application of the 4th test run led to concordant and unambiguous results.

Minimal to moderate compound residues remained on the tissues and / or the inner wall of the plastic inserts after the washing procedure in all test runs.

Overall, the viability values in most tissues (especially in tissues in test run 4) are well above the cut off for skin irritation. Thus, it is concluded that the test substance does not indicate an irritation potential.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Mar - Apr 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
09 Oct 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 0017319511
- Expiration date of the batch: 18 Dec 2018
- Purity: > 99 %
- pH value: ca. 6
- Physical state / color: Solid, waxy / white to yellowish

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: To improve application, the solid waxy test substance was heated at about 50 °C for about 15 minutes. Before application, the test substance was cooled down to room temperature.
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Alzey, Emil Färber GmbH & Co. KG, Robert-Bosch-Straße 23, 55232 Alzey, Germany
- Characteristics of donor animals (e.g. age, sex, weight): min. 12 months and max. 60 months old
- indication of any existing defects or lesions in ocular tissue samples: no
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
Duration of treatment / exposure:
4 hours
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Bovine eyes are obtained as a by-product of freshly slaugthered cattle (age of the animals: minimum 12 months, maximum 60 months).

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle's MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period, the medium in both chambers was replaced by fresh pre-warmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 556 opacity units were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups. Each corneal holder was uniquely identified with a number on the chambers.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
deionized water

POSITIVE CONTROL USED
Imidazole (CAS-No. 288-32-4) 20 % (w/v) solution in deionized water for non-surfactant solid test substances

APPLICATION DOSE AND EXPOSURE TIME
750 µL for 4 hours

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The NC and the PC were removed from the anterior chamber by using a syringe and the epithelium was washed at least 3 times with Eagle's MEM (containing phenol red) and once with Eagle's MEM (without phenol red). Because the test substance could not be removed by using a syringe the epithelium was rinsed with the open chamber method.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Before measurement, each cornea was visually observed and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: decision criteria according to the OECD Guideline 437 (adopted 09 Oct 2017)
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
9.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: Opacity score of the test substance, the NC and the PC

Test substance identification

Cornea-No.

Initial opacity

Final opacity

Opacity change

Corrected opacity change*

Mean

SD

Test substance

7

8

9

3.2

4.0

4.9

8.9

8.2

8.5

5.7

4.2

3.5

0.0

0.0

0.0

0.0

0.0

NC

1

2

3

4.5

5.5

7.0

15.2

12.9

9.1

10.7

7.5

2.1

NA

NA

NA

6.7

4.3

PC

4

5

6

4.1

4.9

2.9

104.8

96.5

113.3

100.7

91.5

110.4

93.9

84.8

103.7

94.1

9.5

* negative values are set to zero for further calculation

Table 2: Permeability score of the test substance, the NC and the PC

Test substance identification

Cornea-No.

Mean OD490

Dilution factor

Mean corrected OD490

Mean

SD

Test substance

7

8

9

0.929

0.447

0.581

1

1

1

0.925

0.443

0.577

0.648

0.249

NC

1

2

3

0.005

0.004

0.003

1

1

1

NA

NA

NA

0.004

0.001

PC

4

5

6

0.266

0.491

0.345

5

5

5

1.328

2.449

1.721

1.833

0.569

Table 3: In Vitro Irritancy Score (IVIS) of the test substance, the NC and the PC

Test substance identification

Cornea-No.

Opacity per cornea*

Permeability per cornea

IVIS

Per cornea

Per group

Mean

SD

Test substance

7

8

9

0.0

0.0

0.0

0.925

0.443

0.577

13.9

6.6

8.7

9.7

3.7

NC

1

2

3

10.7

7.5

2.1

0.005

0.004

0.003

10.7

7.5

2.1

6.8

4.3

PC

4

5

6

93.9

84.8

10.37

1.328

2.449

1.721

113.9

121.5

129.5

121.6

7.8

* negative values are set to zero for further calculation

Interpretation of results:
other: no severe eye irritant/no eye damage
Conclusions:
No prediction can be made for eye irritation according to GHS criteria based on the results of this in vitro study alone.
Executive summary:

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 µL undiluted test substance to the epithelial surface of isolated bovine corneas.

The solid, waxy test substance could not be homogeneously prepared as a 20 % preparation in deionized water. Therefore, a piece of Parafilm (matched to the size of the application area) was covered with 750 µL of the undiluted (at ca. 50 °C heated) test substance and was applied directly to the epithelial surface of the cornea. Before application, the test substance was cooled down to room temperature. Three corneas were treated with the test substance for an exposure period of 4 hours. In addition to the test substance, a negative control (NC; deionized water) and a positive control (PC; 20 % imidazole in deionized water) were applied to three corneas each. Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. The following results were obtained in the BCOP Test:

Table 1: Mean values for opacity, permeability and IVIS of the test substance, the NC and the PC

Test substance identification

Mean Opacity Value

Mean Permeability Value

Mean In Vitro Irritancy Score

Test substance

0.0

0.648

9.7

NC

6.7

0.004

6.8

PC

94.1

1.833

121.6

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Mar - Apr 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
09 Oct 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 0017319511
- Expiration date of the batch: 18 Dec 2018
- Purity: > 99 %
- pH value: ca. 6
- Physical state / color: solid, waxy / white to yellowish

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: To improve application, the solid waxy test substance was heated at about 50 °C for about 15 minutes. Before application, the test substance was cooled down to room temperature.
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability : The test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short term topical exposure. The test is designed to predict an eye irritation potential of a chemical by using the three-dimensional, human cornea model EpiOcular. After application of the test material to the surface of the EpiOcular tissue, the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol extraction of the formazan from the tissues, the optical density of the extract is spectrophotometrically determined. The optical density of the extracts of the tissues treated with the test substance is compared to values from negative control tissues and expressed as relative tissue viability.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular model (OCL-200) is a three-dimensional, non-keratinized tissue construct composed of normal human-derived, epidermal keratinocytes used to model the human corneal epithelium. The EpiOcular tissues (surface 0.6 cm²) are cultured on cell culture inserts and are commercially available as kits containing 24 tissues on shipping agarose.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
other: MTT reduction control (KC): sterile deionized water or test substance
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2 tissues per test run and test group
Details on study design:
- Details of the test procedure used : The test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short term topical exposure. The test is designed to predict an eye irritation potential of a chemical by using the three-dimensional, human cornea model EpiOcular. After application of the test material to the surface of the EpiOcular tissue, the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol extraction of the formazan from the tissues, the optical density of the extract is spectrophotometrically determined. The optical density of the extracts of the tissues treated with the test substance is compared to values from negative control tissues and expressed as relative tissue viability.
- RhCE tissue construct used, including batch number : OCL-200 tissue, Lot No. 27027 (test run 1) and 27031 (test run 2)
- Doses of test chemical and control substances used : 50 µL of undiluted test substance, deionized water (NC) and neat methyl acetate (PC), respectively
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : 6 hours exposure time at 37 °C and 18 hours post-incubation at 37 °C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : freeze-killed control tissues were treated with the test article and the negative control each in the same way
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : Sunrise Absorbance Reader; For the determination of the optical density of colored extracts. Measurement using a filter wavelength 570 nm without reference filter.
- Description of the method used to quantify MTT formazan : The formazan that was metabolically produced by the tissues was extracted by overnight incubation of the tissues in isopropanol at room temperature or by incubation for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was spectrophotometrically determined. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :: The OD570 values determined for the various tissues are measures of their viability. The ratio of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether a test material was an irritant. The OD570 value measured and corrected for each individual tissue was calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way was calculated. In case of direct MTT reduction by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the tissues treated with the test substance (mean KC-corrected OD570). Since killed tissues might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero, it is subtracted from the respective mean OD570 to result in the mean KC-corrected OD570. The mean KC-corrected OD570 represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance. The quantification of tissue viability is presented as ratio of the mean OD570 (or mean KC-corrected OD570, if applicable) divided by the respective OD570 NC value in percent.
- Acceptable variability between tissue replicates for positive and negative controls: The absolute OD570 of the NC tissues in the MTT test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is > 0.8. The mean OD570 of the NC should not exceed 2.5. Methyl acetate used as PC usually leads to a tissue viability of approx. 25 %. A viability of < 50 % is acceptable.
- Acceptable variability between tissue replicates for the test chemical: Two tissues were treated under the same conditions. A variability between the two tissues is considered to be acceptable if the relative difference of the viability is < 20 %.
Irritation parameter:
other: % tissue viability
Run / experiment:
1st test run
Value:
9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: % tissue viability
Run / experiment:
2nd test run
Value:
4.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: 1st test run: Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

Test substance identification

 

 

Tissue 1

Tissue 2

Mean

Inter-tissue variability [%]

NC

Viable tissues

Mean OD570

0.907

0.914

0.910

 

Viability [% of NC]

99.6

100.4

100.0

0.7

KC tissues

Mean OD570

0.022

0.022

0.022

 

Viability [% of NC]

2.4

2.4

2.4

0.1

Test substance

Viable tissues

Mean OD570

0.089

0.193

0.141

 

Viability [% of NC]

9.8

21.1

15.5

11.4

KC tissues

Mean OD570

KC NC corrected

0.071

0.046

0.059

 

Viability [% of NC]

7.8

5.1

6.4

2.7

Finale relative mean viability of tissues after KC correction [% of NC]

9.0

 

PC

Viable tissues

Mean OD570

0.268

0.250

0.259

 

Viability [% of NC]

29.4

27.4

28.4

2.0

Table 2: 2nd test run: Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

Test substance identification

 

 

Tissue 1

Tissue 2

Mean

Inter-tissue variability [%]

NC

Viable tissues

Mean OD570

1.987

2.009

1.998

 

Viability [% of NC]

99.4

100.6

100.0

1.1

KC tissues

Mean OD570

0.025

0.025

0.025

 

Viability [% of NC]

1.3

1.2

1.3

0.0

Test substance

Viable tissues

Mean OD570

0.204

0.055

0.130

 

Viability [% of NC]

10.2

2.8

6.5

7.4

KC tissues

Mean OD570

KC NC corrected

0.031

*

0.031

 

Viability [% of NC]

1.5

*

1.5

 

Finale relative mean viability of tissues after KC correction [% of NC]

4.9

 

PC

Viable tissues

Mean OD570

0.440

0.297

0.369

 

Viability [% of NC]

22.0

14.9

18.5

7.2

* KC tissue got lost during the washing procedure. Therefore, evaluation is based on the result of a single KC tissue. Since all other quality criteria of the test weremet and due to the unambiguous results, this deviation is not considered to adversely affect the evaluation of this study.

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Based on the overall available results, the substance cause eye irritation.
Executive summary:

The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50 µL undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular).

The test substance could not be homogeneously distributed on the whole application area. Therefore, a metal pin was covered with 50 µL undiluted (at ca. 50 °C heated) test substance and was applied covering the whole tissue surface. Before application the test substance was cooled down to room temperature. Two test runs were performed. Two EpiOcular tissues per test run were incubated with the test substance for 6 hours followed by an 18 hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure / post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the peidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The following results were obtained in the EpiOcular eye irritation assay:

The test substance is able to directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced.

Results of the 1st test run: The final relative mean viability of the tissues treated with the test substance was 9.0 %. The tissue viability (mean OD570) of the negative control was exceptionally low and lies out of range of the historical data. Thus, a 2nd test run was conducted to verify the results.

Results of the 2nd test run: The final relative mean viability of the tissues treated with the test substance of the 2nd test run was 4.9 %. All acceptance criteria were met.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation in vitro turnkey testing strategy

The objective was to assess the skin corrosion and irritation potential of the test substance. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating / corrosion potential including transport classification. Therefore, three in vitro assays were proposed for this in vitro skin irritation and corrosion test strategy including transport classification: The Skin Corrosion Test (SCT), Skin Irritation Test (SIT) and In vitro Membrane Barrier Test (Corrositex). However, in the current case, the results derived with SIT and SCT alone were sufficient for a final assessment. Therefore, further testing in Corrositex was waived. Four test runs of the SIT were performed, due to high variability of results.

The potential of the test substance to cause dermal corrosion / irritation was assessed by a single topical application of 50 µL (corrosion test) or 30 µL (irritation test) undiluted test substance to a reconstructed three-dimensional, human epidermis model (EpiDerm). The test substance could not be homogeneously distributed on the whole application area. Therefore, a metal pin covered with 50 µL (SCT) or 30 µL (SIT) undiluted, at ca. 50 °C heated test substance was applied covering the whole tissue surface. Before application, the test substance was cooled down to room temperature. Due to technical issues at removal of the waxy test substance during several test runs of the skin irritation test, the tissues were wetted with 25 µL sterile PBS before application of the test substance in a 4th test run. For the corrosion test, two EpiDerm tissues were incubated with the test substance for 3 minutes and 1 hour each. The irritation test was performed with three EpiDerm tissues per test run, which were incubated with the test substance for 1 hour followed by a 42 hour post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure / post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

Results of the Corrosion Test (SCT):

The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 104.8 %, and it was 59.3 % after an exposure period of 1 hour.

Results of the Irritation Test (SIT):

Four test runs were performed, due to high variability of results.

1st test run: Due to the non-concordant replicate measurements (relative tissue viability) of the test substance treated tissues (values for single tissues (without KC correction): 63.6 %, 29.2 % and 77.1 %), another test run of the EpiDerm skin irritation test was performed to clarify the result.

2nd test run: Again non-concordant replicate measurements (relative tissue viability) of the test substance treated tissues (values for single (without KC correction): 67.1 %, 30.1 % and 65.6 %) were obtained. The 2nd tissue was partly detached during the washing procedure. Thus, another test run of the EpiDerm skin irritation test was performed.

3rd test run: The replicate measurements (relative tissue viability) of the test substance treated tissues were again not concordant (values for single tissues (without KC correction): 60.5 %, 25.1 % and 45.7 %).

The non-concordant results of test run 1 to 3 of the EpiDerm skin irritation test may be attributed to technical issues at removal of the waxy test substance. Thus, the tissues were wetted with 25 µL sterile PBS before application of the test substance in a 4th test run.

4th test run: The final relative mean viability of the tissues treated with the test substance of the 4th test run was 66.7 % (values for single tissues (without KC correction): 69.5 %, 76.2 % and 68.8 %). All acceptance criteria were met. Wetting the tissues prior to application of the 4th test run led to concordant and unambiguous results.

Minimal to moderate compound residues remained on the tissues and / or the inner wall of the plastic inserts after the washing procedure in all test runs. Overall, the viability values in most tissues (especially all tissues in test run 4) are well above the cut off for skin irritation. Thus, it is concluded that the test substance does not indicate an irritation potential.

Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm in vitro skin irritation and corrosion test strategy including transport classification under the test conditions chosen.

Eye irritation in vitro turnkey testing strategy

The objective was to assess the eye irritating potential of the test substance. By using the methods currently available a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

BCOP

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 µL undiluted test substance to the epithelial surface of isolated bovine corneas.

The solid, waxy test substance could not be homogeneously prepared as a 20 % preparation in deionized water. Therefore, a piece of Parafilm (matched to the size of the application area) was covered with 750 µL of the undiluted (at ca. 50 °C heated) test substance and was applied directly to the epithelial surface of the cornea. Before application, the test substance was cooled down to room temperature. Three corneas were treated with the test substance for an exposure period of 4 hours. In addition to the test substance, a negative control (NC; deionized water) and a positive control (PC; 20 % imidazole in deionized water) were applied to three corneas each. Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. The following results were obtained in the BCOP Test:

Table 1: Mean values for opacity, permeability and IVIS of the test substance, the NC and the PC

Test substance identification

Mean Opacity Value

Mean Permeability Value

Mean In Vitro Irritancy Score

Test substance

0.0

0.648

9.7

NC

6.7

0.004

6.8

PC

94.1

1.833

121.6

EpiOcular

The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50 µL undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular).

The test substance could not be homogeneously distributed on the whole application area. Therefore, a metal pin was covered with 50 µL undiluted (at ca. 50 °C heated) test substance and was applied covering the whole tissue surface. Before application the test substance was cooled down to room temperature. Two test runs were performed. Two EpiOcular tissues per test run were incubated with the test substance for 6 hours followed by an 18 hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure / post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the peidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The following results were obtained in the EpiOcular eye irritation assay:

The test substance is able to directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced.

Results of the 1st test run: The final relative mean viability of the tissues treated with the test substance was 9.0 %. The tissue viability (mean OD570) of the negative control was exceptionally low and lies out of range of the historical data. Thus, a 2nd test run was conducted to verify the results.

Results of the 2nd test run: The final relative mean viability of the tissues treated with the test substance of the 2nd test run was 4.9 %. All acceptance criteria were met.

Summary of the individual results of the in vitro eye irritation turnkey testing strategy

Table 2: Summary of individual test results and test strategy evaluation

Test method

Test result

Test evaluation

Evaluation test strategy

BCOP Test

Mean IVIS: 9.7

Not identified as corrosive or severe irritant

Ocular irritant

EpiOcular

Final relative mean viabilities per test run:

9.0 % (1sttest run)

4.9 % (2ndtest run)

Irritant

Based on the results of the BCOP and EpiOcular Tests and by applying the evaluation criteria, it was concluded that the test substance shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. A GLP-compliant in vitro turnkey testing strategy is available for skin irritation and eye irritation, respectively. In the skin irritation in vitro turnkey testing strategy, the scores for the test item treated tissues were below the thresholds for classification as an irritant. In the eye irritation in vitro turnkey testing strategy, the test substance showed an eye irritation potential. As a result, the substance is not considered to be classified for skin irritation but for eye irritation Cat. 2 under Regulation (EC) 1272/2008, as amended for the tenth time in Regulation (EC) No. 2017/776.