Registration Dossier

Administrative data

Description of key information

Skin irritation

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 SIT, that were sufficient for a final assessment, the test substance shows no skin irritating potential and further testing in SCT was waived.

Eye irritation

A GLP-compliant in vitro eye irritation turnkey testing strategy was conducted in the BCOP according to OECD Guideline 437 and in the EpiOcular according to OECD Guideline 492 to assess the eye irritating potential of the test substance. Based on the results, the test substance causes serious eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May - Aug 2019
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:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt Rheinland-Pfalz
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 17 Jan 2020
- Physical state / color: Solid / white
- pH value: ca. 5 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, under N2

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For better handling, the test substance was ground with a pestle and mortar.
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): 28696
- Date of initiation of testing: 10.05.2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 28 min at room temperature and 32 min in the incubator
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: one washing step
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mL MTT solution
- Incubation time: 3 h
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm without reference filter
- The test substance is not able to reduce MTT directly, which was demonstrated in a pretest. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was not introduced.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze-killed
- N. of replicates: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-irritant to skin if the viability after exposure and post-treatment incubation is greater than 50 %
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL bulk volume (about 19 mg)

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 % in water
Duration of treatment / exposure:
1 h
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st test run
Value:
96.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2nd test run
Value:
94.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3rd test run
Value:
76
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Table 2: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficients of variation

Test substance identification

 

Tissue 1

Tissue 2

Tissue 3

Mean

SD

CV [%]

NC

Mean OD570

1.687

1.812

1.687

1.729

 

 

Viability

[% of NC]

97.6

104.8

97.6

100.0

4.2

4.2

Test substance

Mean OD570

1.661

1.638

1.315

1.538

 

 

Viability

[% of NC]

96.1

94.8

76.0

89.0

11.2

12.6

PC

Mean OD570

0.059

0.061

0.068

0.062

 

 

Viability

[% of NC]

3.4

3.5

3.9

3.6

0.3

7.3

Table 3: Historic control data of NC and PC of skin irritation test

Historic Range of NC

OD570

 

 

 

 

Period

Mean OD

SD

Mean + 2 SD

Mean – 2 SD

Jan 2017 – Mar 2019

1.831

0.192

2.215

1.447

Historic Range of PC

OD570

 

 

 

 

Period

Mean OD

SD

Mean + 2 SD

Mean – 2 SD

Jan 2017 – Mar 2019

0.048

0.005

0.059

0.037

Relative Viability [%]

Period

Mean %

SD

Mean + 2 SD

Mean – 2 SD

Jan 2017 – Mar 2019

2.6

0.4

3.4

1.9

Interpretation of results:
GHS criteria not met
Conclusions:
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 under the test conditions chosen.
Executive summary:

The objective of the study was to assess the skin irritation and corrosion 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. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

However, in the current case the results derived with SIT alone were sufficient for a final assessment. Therefore, further testing in SCT was waived.

The potential of the test substance to cause dermal irritation was assessed by a single topical application of ca. 25 μL bulk volume (about 19 mg) undiluted ground test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). Before application the epidermis surface was moistend with 25 μL sterile PBS. Thereafter, the solid ground test material was applied directly onto the tissue surface and distributed together with the fluid, so that the surface of the epidermis model was uniformly and completely covered.

The Skin Irritation Test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. In addition to the test substance, 30 μL / tissue of a negative control (PBS) and a positive control (5% SDS) were applied to three tissues each.

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 following results were obtained in the EpiDerm™ Skin Irritation Test (SIT):

The test substance is not able to reduce MTT directly, which was demonstrated in a pretest. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was not introduced.

The relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 89%.

Application of the positive control 5% SDS showed a relative mean viability of the tissues of 3.6% and reflects the expected sensitivity of the tissues. The mean OD570 of the negative control (PBS) fulfill the acceptance criteria and demonstrate the validity of the assay.

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 under the test conditions chosen.

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:
May - Aug 2019
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:
09 Oct 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt Rheinland-Pfalz
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 17 Jan 2020
- Physical state / color: Solid / white
- pH value: Ca. 5 (undiluted test substance moistened with deionized water, determined in the lab prior to the start of the GLP study); Ca. 5 (20 % aqueous preparation; determined in the lab prior to start of the GLP study)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, under N2
- Solubility and stability of the test substance in the solvent/vehicle: After stirring with a magnetic stirrer, the test substance was soluble in the vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance preparation was produced on a weight per volume (w/v) basis shortly before application. After stirring with a magnetic stirrer, the test substance was soluble in the vehicle.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : 20 % (w/v) solution in deionized water
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 animals
- indication of any existing defects or lesions in ocular tissue samples: Corneas free of defects (opacity, scratches, pigmentation etc.)
Vehicle:
water
Remarks:
deionized
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20 % (w/v) in deionized water

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
Duration of treatment / exposure:
4 h
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF 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 consist 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.

QUALITY CHECK OF THE ISOLATED CORNEAS : 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 < 561 opacity units were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : deionized water

POSITIVE CONTROL USED : 20 % solution (w/v) of imidazole in deionized water

APPLICATION DOSE AND EXPOSURE TIME : 750 µL, 4 h

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: one

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 microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: see table 1
Irritation parameter:
in vitro irritation score
Run / experiment:
1st experiment
Value:
65
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
2nd experiment
Value:
45.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
3rd experiment
Value:
82.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Table 2: 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

63.9

42.8

73.5

0.078

0.171

0.584

65.0

45.3

82.3

64.2

18.5

NC

1

2

3

2.8

9.8

8.5

0.004

0.005

0.008

2.9

9.9

8.6

7.1

3.7

PC

4

5

6

91.0

96.7

88.8

1.606

2.043

2.389

115.1

127.3

124.6

122.4

6.4

Table 3: 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

6.5

5.2

6.5

77.4

55.0

87.1

70.9

49.8

80.5

63.9

42.8

73.5

60.0

15.7

NC

1

2

3

6.9

6.0

6.4

9.7

15.8

14.9

2.8

9.8

8.5

NA

NA

NA

7.0

3.7

PC

4

5

6

4.9

6.6

5.6

103.0

110.3

101.4

98.1

103.7

95.8

91.0

96.7

88.8

92.2

4.1

Table 4: 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

0.084

0.176

0.590

1

1

1

0.078

0.171

0.584

0.278

0.270

NC

1

2

3

0.004

0.006

0.008

1

1

1

NA

NA

NA

0.006

0.002

PC

4

5

6

0.322

0.410

0.479

5

5

5

1.606

2.043

2.389

2.013

0.393

Table 5: Historic control data of the BCOP Test

Historic Range of NC (protocol for solids)

Historic period

Jan 2017 – Mar 2019 (No. of tests performed: 12)

Opacity

Mean

SD

Mean + 2 SD

Mean – 2 SD

9.2

3.7

16.5

1.9

Permeability (OD490)

Mean

SD

Mean + 2 SD

Mean – 2 SD

0.003

0.002

0.006

0.000

In Vitro Irritation Score (IVIS)

Mean

SD

Mean + 2 SD

Mean – 2 SD

9.2

3.7

16.6

1.9

Historic Range of PC (20 % Imidazole)

Historic period

Jan 2017 – Mar 2019 (No. of tests performed: 9)

Opacity

Mean

SD

Mean + 2 SD

Mean – 2 SD

93.6

9.9

113.5

73.7

Permeability (OD490)

Mean

SD

Mean + 2 SD

Mean – 2 SD

2.234

0.578

3.389

1.078

In Vitro Irritation Score (IVIS)

Mean

SD

Mean + 2 SD

Mean – 2 SD

127.1

13.6

154.2

100.0

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In the BCOP Test the mean IVIS (64.2) of the corneas treated with the test substance indicated a serious eye irritation potential (BCOP cut-off threshold 55) of the test substance.
However, some variability between the results of the individual corneas treated with the test substance was noted and the mean IVIS lies minimal below the borderline range (IVIS 45.0 to 65.0) for evaluation.
Nevertheless, the result of the BCOP has to be considered to indivate ocular corrosion or severe irritation of the test substance, because the IVIS values of two corneas and the mean value of all corneas lie well above the BCOP cut-off threshold of 55 and the mean IVIS lies minimal below the upper value of the borderline range.
Executive summary:

The potential of the test substance to cause severe ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL 20% test-substance preparation to the epithelial surface of isolated bovine corneas.

Three corneas were treated with the test-substance preparation for an exposure period of 4 hours.

Besides 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 measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively 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 mean IVIS (64.2) of the corneas treated with the 20% test substance preparation indicated serious eye damage (BCOP cut-off threshold 55) of the test substance.

The results of the test substance, the negative control (deionized water) and the positive control (20% imidazole) fulfilled the acceptance criteria and demonstrate the validity of the assay.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May - Aug 2019
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:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt Rheinland-Pfalz
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 17 Jan 2020
- Physical state / color: Solid / white
- pH value: Ca. 5 (undiluted test substance moistened with deionized water, determined in the lab prior to the start of the GLP study); Ca. 5 (20 % aqueous preparation; determined in the lab prior to start of the GLP study)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, under N2

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was applied undiluted. Therefore, no preparation of the test substance in a vehicle was performed.

FORM AS APPLIED IN THE TEST (if different from that of starting material): undiluted
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability : The EpiOcularTM 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 EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200) containing 24 tissues on shipping agarose.
- Description of the cell system used: OCL-200 (Tissue Lot Number 30609), Supplier: MatTek In Vitro Life Sciences Laboratories, Bratislava, Slovakia
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 µL
Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
18 h
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used
- RhCE tissue construct used, including batch number : OCL-200 (Tissue Lot Number 30609)
- Doses of test chemical and control substances used : 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : 6 h exposure in the incubator, 18 h post-incubation in the incubator
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : freeze-killed control tissues (KC)
- 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) : 570 nm
- Description of the method used to quantify MTT formazan : The optical density at a wavelength of 570 nm of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wellsfilled 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 irritation potential of the test material is predicted from the mean relative tissue viabilities compared to the negative control tissues treated concurrently with sterile water. A chemical is considered as "non-irritant” (no UN GHS Category) if the mean relative tissue viability with a test material is greater than 60%. A single test composed of at least two tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in case of borderline results such as non-concordant replicate measurements and/or mean percent tissue viability equal to ± 5% of the cut-off value, a second test should be considered as well as a third one in case of discordant results between the first two tests.
Irritation parameter:
other: mean viability [%]
Run / experiment:
mean
Value:
2.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 2: 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

Mean OD570

1.305

1.425

1.365

 

Viability [% of NC]

95.6

104.4

100.0

8.8

Test substance

Mean OD570

0.030

0.029

0.029

 

Viability [% of NC]

2.2

2.1

2.1

0.0

PC

Mean OD570

0.345

0.254

0.299

 

Viability [% of NC]

25.3

18.6

21.9

6.7

Table 3: Historic control data of the EpiOcular Test

Historic Range of NC

OD570

Protocol

Period

Mean OD

SD

Mean + 2 SD

Mean – 2 SD

Protocol for liquids

Jan 2017 – Apr 2019

1.940

0.476

2.891

0.988

Protocol for solids

Jan 2017 – Mar 2019

1.867

0.387

2.640

1.093

Historic Range of PC

OD570

Protocol

Period

Mean OD

SD

Mean + 2 SD

Mean – 2 SD

Protocol for liquids

Jan 2017 – Apr 2019

0.558

0.173

0.904

0.212

Protocol for solids

Jan 2017 – Mar 2019

0.336

0.109

0.554

0.119

Relative Viability [%]

Protocol

Period

Mean %

SD

Mean + 2 SD

Mean – 2 SD

Protocol for liquids

Jan 2017 – Apr 2019

29.2

8.2

45.7

12.8

Protocol for solids

Jan 2017 – Mar 2019

18.0

4.6

27.2

8.8

Interpretation of results:
study cannot be used for classification
Conclusions:
In the EpiOcular Test, the test substance was assessed to have an eye irritation potential (relative mean viability of the tissues treated with the test substance was 2.1 %).
Executive summary:

The potential of the test substance to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (about 26 mg) undiluted ground test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™), so that the surface of the cornea model was completely covered with the test substance.

Two EpiOcular™ tissues were pretreated with 20 μL PBS in order to wet the tissue surface and then incubated with the test substance for 6 hours followed by an 18-hour post-incubation period.

In addition to the test substance, 50 μL per tissue of a negative control (deionized water) and a positive control (methyl acetate) were applied to two tissues each.

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 ratio of the values indicates the relative tissue viability.

The following results were obtained in the EpiOcular™ Eye Irritation Test:

The test substance is not able to reduce MTT directly, which was demonstrated in a pretest. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was not introduced.

The relative mean viability of the tissues treated with the test substance was 2.1%. The variability between the results of the tissues are within the acceptance range.

Application of the positive control methyl acetate showed a relative mean viability of the tissues of 21.9% and reflects the expected sensitivity of the tissues

The mean OD570 of the negative control (deionized water) fulfill the acceptance criteria and demonstrate the validity of the assay.

In the EpiOcular Test, the test substance was assessed to have an eye irritation potential. However, no prediction of the eye irritation potential can be made. Thus, the BCOP assay was conducted afterwards for final assessment.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

The objective of the study was to assess the skin irritation and corrosion 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. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

However, in the current case the results derived with SIT alone were sufficient for a final assessment. Therefore, further testing in SCT was waived.

The potential of the test substance to cause dermal irritation was assessed by a single topical application of ca. 25 μL bulk volume (about 19 mg) undiluted ground test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). Before application the epidermis surface was moistend with 25 μL sterile PBS. Thereafter, the solid ground test material was applied directly onto the tissue surface and distributed together with the fluid, so that the surface of the epidermis model was uniformly and completely covered.

The Skin Irritation Test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. In addition to the test substance, 30 μL / tissue of a negative control (PBS) and a positive control (5% SDS) were applied to three tissues each.

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 following results were obtained in the EpiDerm™ Skin Irritation Test (SIT):

The test substance is not able to reduce MTT directly, which was demonstrated in a pretest. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was not introduced.

The relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 89%.

Application of the positive control 5% SDS showed a relative mean viability of the tissues of 3.6% and reflects the expected sensitivity of the tissues

The mean OD570 of the negative control (PBS) fulfill the acceptance criteria and demonstrate the validity of the assay.

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 under the test conditions chosen.

Eye Irritation

The objective was to assess the eye irritation 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 irritation 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 the EpiOcular™ Eye Irritation Test.

The potential of the test substance to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (about 26 mg) undiluted ground test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™), so that the surface of the cornea model was completely covered with the test substance.

Two EpiOcular™ tissues were pretreated with 20 μL PBS in order to wet the tissue surface and then incubated with the test substance for 6 hours followed by an 18-hour post-incubation period.

In addition to the test substance, 50 μL per tissue of a negative control (deionized water) and a positive control (methyl acetate) were applied to two tissues each.

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 ratio of the values indicates the relative tissue viability.

The following results were obtained in the EpiOcular™ Eye Irritation Test:

The test substance is not able to reduce MTT directly, which was demonstrated in a pretest. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was not introduced.

The relative mean viability of the tissues treated with the test substance was 2.1%. The variability between the results of the tissues are within the acceptance range.

Application of the positive control methyl acetate showed a relative mean viability of the tissues of 21.9% and reflects the expected sensitivity of the tissues

The mean OD570 of the negative control (deionized water) fulfill the acceptance criteria and demonstrate the validity of the assay.

In the EpiOcular Test, the test substance was assessed to have an eye irritation potential. However, no prediction of the eye irritation potential can be made. Thus, the BCOP assay was conducted afterwards for final assessment.

The potential of the test substance to cause severe ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL 20% test-substance preparation to the epithelial surface of isolated bovine corneas.

Three corneas were treated with the test-substance preparation for an exposure period of 4 hours.

Besides 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 measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively 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 mean IVIS (64.2) of the corneas treated with the 20% test substance preparation indicated serious eye damage (BCOP cut-off threshold 55) of the test substance.

The results of the test substance, the negative control (deionized water) and the positive control (20% imidazole) fulfilled the acceptance criteria and demonstrate the validity of the assay.

Based on the results of the BCOP and EpiOcular Tests and by applying the evaluation criteria, it was concluded that the test substance causes serious eye damage 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) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. A GLP-compliant turnkey testing strategy including the EpiDerm skin corrosion test (SCT, OECD Guideline 431) and the EpiDerm skin irritation test (SIT, OECD Guideline 439) is available for skin irritation. Based on the results of the skin irritation test, the test substance is a non-corrosive. Therefore, it can be concluded that the test item is not considered to be classified for skin irritation under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. A GLP-compliant turnkey testing strategy including the BCOP (OECD Guideline 437) and the EpiOcular (OECD Guideline 492) is available for eye irritation. Based on the results of the the tests, the test substance causes serious eye damage. Therefore, it can be concluded that the test item is considered to be classified for eye irritation Cat. 1 under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.