Registration Dossier

Administrative data

Description of key information

A GLP-compliant in vitro skin irritation turnkey strategy was used to assess the skin irritating potential of the test substance. Both, the skin corrosion test (according to OECD Guideline 431) and the skin irritation test (according to OECD Guideline 439), were negative. Therefore, the substance is not considered to be classified for skin irritation.

A GLP-compliant in vitro eye irritation turnkey strategy was used to assess the eye irritating potential of the test substance. According to the results of the BCOP (OECD Guideline 437) the substance is not classified in UN GHS Category 1. Based on the combined assessment together with the EpiOcular (according to OECD Guideline 492) the substance is considered to be classified for eye irritation UN GHS Category 2.

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 2019
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:
Landesamt für Umwelt Rheinland-Pfalz
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)
- pH value: ca. 4 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study)
- Physical state / color: Solid / yellowish

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

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solid
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number: 28684

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: one washing step with PBS

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

NUMBER OF REPLICATE TISSUES: two tissues per exposure time and test group (12 tissues per test)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze-killed at - 20 °C
- N. of replicates: two killed control tissues per exposure time were treated with the test substance and the negative control, respectively
- Method of calculation used: 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 corrected OD570 KC). 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 corrected OD570 KC. The mean corrected OD570 KC represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one

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): 25 µL

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 N
Duration of treatment / exposure:
3 min and 1 h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure period
Value:
95.5
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:
1 h exposure period
Value:
105.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Table 2: Summary of results after 3 minutes exposure period

Test substance identification

 

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Viable tissues

Mean OD570

2.016

1.996

2.006

 

 

Viability [% of NC]

100.5

99.5

100.0

0.7

0.7

KC tissues

Mean OD570

0.114

0.092

0.103

 

 

Viability [% of NC]

5.7

4.6

5.1

0.8

15.4

Test substance

Viable tissues

Mean OD570

1.995

1.849

1.922

 

 

Viability [% of NC]

99.5

92.2

95.8

5.1

5.4

KC tissues *

Mean OD570

KC NC corrected

0.011

0.000

0.006

 

 

Viability [% of NC]

0.56

0.00

0.28

 

 

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

95.5

PC

Viable tissues

Mean OD570

0.254

0.216

0.235

 

 

Viability [% of NC]

12.7

10.8

11.7

1.3

11.4

*negative values are set to zero for further calculation

Table 3: Summary of results after 1 hour exposure period

Test substance identification

 

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Viable tissues

Mean OD570

1.854

1.875

1.864

 

 

Viability [% of NC]

99.4

100.6

100.0

0.8

0.8

KC tissues

Mean OD570

0.085

0.092

0.088

 

 

Viability [% of NC]

4.5

4.9

4.7

0.3

5.6

Test substance

Viable tissues

Mean OD570

1.941

2.030

1.986

 

 

Viability [% of NC]

104.1

108.9

106.5

3.4

3.2

KC tissues

Mean OD570

KC NC corrected

0.023

0.019

0.021

 

 

Viability [% of NC]

1.2

1.0

1.1

0.1

11.9

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

105.4

PC

Viable tissues

Mean OD570

0.097

0.106

0.101

 

 

Viability [% of NC]

5.2

5.7

5.4

0.3

6.3

Table 4: Historic control data of NC and PC of skin corrosion test

 

 

Exposure Time

Period

Mean

SD

Mean + 2 SD

Mean – 2 SD

NC

OD570

3 minutes

Jan 2017 – Jan 2019

1.728

0.216

2.160

1.295

60 minutes

1.724

0.252

2.227

1.220

PC

3 minutes

0.222

0.071

0.363

0.081

60 minutes

0.097

0.031

0.159

0.036

Relative viability [%]

3 minutes

13.0

4.2

21.3

4.6

60 minutes

5.7

1.8

9.3

2.2

Interpretation of results:
GHS criteria not met
Remarks:
no indication of skin corrosion
Conclusions:
Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin corrosion potential in the EpiDerm™ in vitro skin corrosion test under the test conditions chosen.
Executive summary:

The potential of the test substance to cause dermal corrosion was assessed by a single topical application of ca. 25 μL bulk volume (about 7 mg) undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).

For the skin corrosion test, 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/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 corrosion test:

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 95.5%, and it was 105.4% after an exposure period of 1 hour.

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

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Feb 2019
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:
Landesamt für Umwelt Rheinland-Pfalz
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)

- pH value: ca. 4 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study)
- Physical state / color: Solid / yellowish

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

FORM AS APPLIED IN THE TEST (if different from that of starting material): solid
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number: 28684

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: two washing steps with PBS

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

NUMBER OF REPLICATE TISSUES: three tissues were treated 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): freeze-killed at - 20 °C
- N. of replicates: three
- Method of calculation used: 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 corrected OD570 KC. The mean corrected OD570 KC represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance 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 %.
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): 25 µL

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 %
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure period
Value:
98.1
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 and standard deviations

Test substance identification

 

 

Tissue 1

Tissue 2

Tissue 3

Mean

SD

NC

Viable tissues

Mean OD570

2.142

2.053

2.180

2.125

 

Viability [% of NC]

100.8

96.6

102.6

100.0

3.1

KC tissues

Mean OD570

0.046

0.042

0.042

0.043

 

Viability [% of NC]

2.2

2.0

2.0

2.0

0.1

Test substance

Viable tissues

Mean OD570

2.057

2.072

2.138

2.089

 

Viability [% of NC]

96.8

97.5

100.6

98.3

2.0

KC tissues

Mean OD570

KC NC corrected

0.0045

0.0045

0.0015

0.0035

 

Viability [% of NC]

0.21

0.21

0.07

0.16

0.08

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

98.1

PC

Viable tissues

Mean OD570

0.045

0.046

0.045

0.045

 

Viability [% of NC]

2.1

2.2

2.1

2.1

0.0

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

 

 

Period

Mean

SD

Mean + 2 SD

Mean – 2 SD

NC

OD570

Jan 2017 – Jan 2019

1.816

0.190

2.195

1.437

PC

0.048

0.005

0.059

0.037

Relative viability [%]

2.7

0.4

3.4

1.9

Interpretation of results:
GHS criteria not met
Remarks:
no indication of skin irritation
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 test under the test conditions chosen.
Executive summary:

The potential of the test substance to cause dermal irritation was assessed by a single topical application of ca. 25 μL bulk volume (about 7 mg) undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).

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 postincubation 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 following results were obtained in the EpiDerm™ skin irritation test:

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 1 hour with an about 42-hour post-incubation was 98.1%.

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 test 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:
Feb - Mar 2019
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:
Landesamt für Umwelt Rheinland-Pfalz
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)
- pH value: ca. 4 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study); ca. 3.5 (20 % aqueous preparation, determined in the lab prior to start of the GLP study)
- Physical state / color: Solid / 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: The test substance preparation was produced on a weight per volume (w/v) basis shortly before application by stirring with a high-speed homogenizer (Ultra-Turrax) and a magnetic stirrer. The homogeneity of the test substance preparation during application was provided by stirring with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : 20 % (w/v) suspension 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): minimum 12 months and maximum 60 months of age
- indication of any existing defects or lesions in ocular tissue samples: corneas free of defects (opacity, scratches, pigmentation etc.) were used
Vehicle:
water
Remarks:
deionized
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20 %

VEHICLE
- 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 : Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : deionized water

POSITIVE CONTROL USED : Imidazole 20 % (w/v) in deionized water

APPLICATION DOSE AND EXPOSURE TIME : 750 µL for 4 hours

TREATMENT METHOD: 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 UV/VIS spectrophotometry (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
- IVIS ≤ 3: no UN GHS Category
- IVIS > 3; ≤ 55: No prediction can be made
- IVIS > 55: UN GHS Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
1st test run
Value:
39.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
2nd test run
Value:
36.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: 1st test run: 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

19

20

21

30.2

41.5

46.9

0.003

0.003

0.000

30.2

41.5

46.9

39.5

8.5

NC

13

14

15

4.3

12.0

24.2

0.000

0.001

0.003

4.3

12.0

24.3

13.5

10.1

PC

16

17

18

51.5

256.0

61.6

3.670

1.982

2.385

106.6

285.8

97.4

163.2

106.2

Table 2: 2nd test run: 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

37.9

37.7

32.5

0.004

0.005

0.000

38.0

37.7

32.5

36.1

3.1

NC

1

2

3

2.3

6.7

2.9

0.002

0.004

0.003

2.3

6.7

2.9

4.0

2.4

PC

4

5

6

96.0

96.5

104.3

1.360

2.012

1.569

116.4

126.7

127.8

123.6

6.3

Table 3: Historic control data of the BCOP Test: Negative control (protocol for solids), Jan 2017 - Jul 2018 (No. of tests performed: 8)

 

Mean

SD

Mean + 2 SD

Mean – 2 SD

Opacity

9.6

3.7

17.0

2.1

Permeability [OD570]

0.003

0.001

0.005

0.001

Table 4: Historic control data of the BCOP Test: Positive control (20 % Imidazole), Jan 2017 - Jul 2018 (No. of tests performed: 8)

 

Mean

SD

Mean + 2 SD

Mean – 2 SD

Opacity

92.9

10.4

113.7

72.1

Permeability [OD570]

2.307

0.571

3.449

1.165

In Vitro Irritation Score (IVIS)

127.5

14.4

156.4

98.7

Interpretation of results:
other: not identified as corrosive or severe irritant
Conclusions:
Based on the results of the BCOP and by applying the evaluation criteria, it was concluded that the test substance was not identified as corrosive or severe irritant under the test conditions chosen. No further assumptions can be made based on 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 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. 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:

Two test runs were performed. The results obtained in the 1st test run did not indicate a severe eye irritation potential of the

test substance. However, high variability between the individual corneas treated with the negative control (NC) and positive control (PC) were noted. Therefore, the study was repeated to verify the result and to meet the acceptance criteria of the test.

The obtained results of the test substance in the 2nd test run of the BCOP test verified the findings of the 1st experiment. All acceptance criteria of the test were fulfilled.

Based on the results of the BCOP and by applying the evaluation criteria, it was concluded that the test substance was not identified as corrosive or severe irritant under the test conditions chosen. No further assumptions can be made based on this in vitro study alone.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Jan 2019
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:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt Rheinland-Pfalz
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)
- pH value: ca. 4 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study); ca. 3.5 (20 % aqueous preparation, determined in the lab prior to start of the GLP study)
- Physical state / color: Solid / yellowish

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

FORM AS APPLIED IN THE TEST (if different from that of starting material): The test substance is applied undiluted.
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 EpiOcularTM. After application of the test material to the surface of the EpiOcularTM 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: 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 (EpiOcular 200) 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): ca. 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
Details on study design:
- RhCE tissue construct used, including batch number : OCL-200, tissue lot no. 27089
- Doses of test chemical and control substances used : 50 µL
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : OCL-200 tissue that is killed by freezing at - 20 °C
- Number of tissue replicates used per test chemical and controls : 2
- Wavelength used for quantifying MTT formazan: 570 nm
- 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 %.
Irritation parameter:
other: tissue viability [%]
Run / experiment:
mean
Value:
40
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: 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

2.284

2.280

2.282

 

Viability [% of NC]

100.1

99.9

100.0

0.2

KC tissues

Mean OD570

0.029

0.031

0.030

 

Viability [% of NC]

1.3

1.4

1.3

0.1

Test substance

Viable tissues

Mean OD570

0.915

0.929

0.922

 

Viability [% of NC]

40.1

40.7

40.4

0.6

KC tissues

Mean OD570

KC NC corrected

0.008

0.013

0.010

 

Viability [% of NC]

0.3

0.6

0.4

0.2

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

40.0

PC

Viable tissues

Mean OD570

0.396

0.337

0.367

 

Viability [% of NC]

17.3

14.8

16.1

2.6

Table 2: Historic control data of the EpiOcular Test

 

Period

Protocol

Mean

SD

Mean + 2 SD

Mean – 2 SD

NC [OD570]

Jan 2017 – Dec 2018

Protocol for liquids

1.901

0.497

2.896

0.906

Jan 2017 – Oct 2018

Protocol for solids

1.822

0.400

2.623

1.021

PC [OD570]

Jan 2017 – Dec 2018

Protocol for liquids

0.536

0.166

0.869

0.203

Jan 2017 – Oct 2018

Protocol for solids

0.326

0.113

0.553

0.100

Relative viability [%]

Jan 2017 – Dec 2018

Protocol for liquids

28.7

7.4

43.5

13.8

Jan 2017 – Oct 2018

Protocol for solids

17.9

4.9

27.7

8.0

Interpretation of results:
other: irritant
Conclusions:
The final relative mean viability of the tissues treated with the test substance was 40 %. The test substance is therefore considered to be classified for eye irritation. No prediction can be made on the UN GHS Category based on this in vitro assay alone.
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 12 mg) undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™).

Two EpiOcular™ tissues were incubated with the test substance for 6 hours followed by a 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 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 assay:

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

The final relative mean viability of the tissues treated with the test substance was 40 %.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

In Vitro Skin Irritation Turnkey Testing Strategy

The objective 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). The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of ca. 25 μL bulk volume (about 7 mg) undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). For the skin corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. 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 postincubation 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 following results were obtained in the EpiDerm™ skin corrosion/irritation test:

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 Skin 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 95.5%, and it was 105.4% after an exposure period of 1 hour.

Results of the Skin Irritation Test (SIT):

The final 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 98.1%.

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.

In Vitro Eye Irritation Turnkey Testing Strategy

The objective was to assess the eye irritating potential of 550Acid. 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 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. 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: Two test runs were performed. The results obtained in the 1st test run did not indicate a severe eye irritation potential of the test substance. However, high variability between the individual corneas treated with the negative control (NC) and positive control (PC) were noted. Therefore, the study was repeated to verify the result and to meet the acceptance criteria of the test. The obtained results of the test substance in the 2nd test run of the BCOP test verified the findings of the 1st experiment. All acceptance criteria of the test were fulfilled.

EpiOcular

The potential of the test substance to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (about 12 mg) undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™).

Two EpiOcular™ tissues were incubated with the test substance for 6 hours followed by a 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 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 assay:

The test substance is able to directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced. The final relative mean viability of the tissues treated with the test substance was 40%.

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) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) 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. Both tests indicate that the substance is negative and therefore not considered to be classified for skin irritation.

A GLP-compliant turnkey testing strategy including the BCOP (OECD Guideline 437) and the EpiOcular (OECD Guideline 492) is available for eye irritation. In the BCOP, the IVIS for the treated tissues indicates that the test substance does not cause irreversible damage to the eyes. The result of the EpiOcular demonstrates that the test substance has to be classified for eye irritation. Therefore, it can be concluded that the test item is considered to be classified for eye irritation Cat. 2 under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.