Registration Dossier

Administrative data

Description of key information

Skin corrosion, in vitro: non-corrosive (3 -minutes viability 93% ; 1 -hour viability 121%), EPIDERM, OECD TG 431, 2019

Skin irritation, in vitro: irritating (15 minutes exposure/42hour incubation viability = 9.4%), EPISKIN, OECD TG 439, 2020

Eye irritation: non-irritating; ICE class I x 3, OECD TG 438, 2020

Eye irritation, in vitro: study result: no-prediction can be made (relative viability = 56% and < 60%), EPIOCULAR EIT, OECD TG 492, 2019

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08-10-2019 to 01-11-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 52%) which does not affect the reliability of the study
Qualifier:
according to
Guideline:
other: EU Method B.40 BIS : In Vitro Skin Corrosion: Human Skin Model Test
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 52%) which does not affect the reliability of the study
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch number(s): Lot no.: 32100
- Production date: Not reported.
- Shipping date: Not reported. Although the CoA for the tissues QA is provided in the full study report.
- Delivery date: 30-10-2019 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: ca. 30-10-2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.0 °C, 5 ± 0.5% CO2 (actual: 36.1 – 36.9 °C)
- Temperature of post-treatment incubation (if applicable): 37 ± 1.0 °C, 5 ± 0.5% CO2; for 3 hours. For further information see below.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 μL DMEM until 6 tissues (= one application time) were dosed and rinsed. For the cell viability measurements: The DMEM was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature. Prior to spectrophotometric analysis for extracted formazan.
- Observable damage in the tissue due to washing: Not applicable.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5.0 mg/mL MTT concentrated diluted (1:5) in MTT diluent solution prepared in assay medium
- Incubation time: For the cell viability measurements: The DMEM was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature. Prior to spectrophotometric analysis for extracted formazan.
- Spectrophotometer: M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: Without reference filter
- Filter bandwidth: No reported.
- Linear OD range of spectrophotometer: Not reported.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Within NC and PC historic control ranges presented within the report.
- Barrier function: See above.
- Morphology: See above.
- Contamination: Not applicable.
- Reproducibility: The positive control had a mean relative tissue viability of 7.7% after the 1-hour exposure. The absolute mean OD570 of the negative control tissues was
within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20-100% viability the Coefficient of Variation between tissue replicates was ≤ 11%, indicating that the test system functioned properly.
- Other: As an additional quality control step, the results of the assay are compared to the historical ranges for negative and positive controls generated by the testing laboratory to confirm the functioning of the test system.

NUMBER OF REPLICATE TISSUES: Two (2), duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: In the previously conducted pretest for direct MTT reduction and colour interference (conducted in separated study cited in full study report): the solution containing the test item was colourless and/or solutions did not turn blue / purple and a blue / purple precipitate was not observed which indicated that the test item did not directly reduce MTT. It was unnecessary to run killed tissues.
- Procedure used to prepare the killed tissues (if applicable): Not applicable.
- N. of replicates : Not applicable.
- Method of calculation used: Not applicable.
- Other: In the assessment of Colour Interference with the MTT endpoint, the solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause colour interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One (n=1) for exposure time 3 minutes and exposure time 1-hour

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%.]
- Cut off points in accordance with OECD TG 431 and the GHS and CLP Classification systems.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: Not applicable.
- Skin corrosion is expressed as the remaining cell viability after exposure to the test substance at exposure times 3-minutes and/or 1-hour with 3-hours incubation. Where necessary, direct MTT reduction, colour interference was completed.

OTHER:
EpiDerm Skin Model (EPI-200, Lot no.: 32100 kit K&J). The test consists of topical application of the test item on the skin tissue for 3-minute and 1-hour. After exposure the skin tissue is thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. The EpiDerm Skin Model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Preincubation:
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM.

Application of test item and rinsing:
The plates were incubated for approximately 3 hours at 37 ± 1.0°C. The medium was replaced with fresh DMEM just before the test item was applied. Two tissues were used for a 3-minute exposure and two for a 1-hour exposure. Test item: 50 µL of the undiluted test item was added into the 6-well plates on top of the skin tissues. Negative Control: similarly treated with 50 µl Milli-Q water only. Positive control: 50 µL 8N KOH (potassium hydroxide 8 normal solution). After the exposure periods (3 minutes and 1 hour, respectively), the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µl DMEM until 6 tissues (= one application time) were dosed and rinsed. All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 59 - 84%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.1 – 36.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on testing laboratory historical data these deviations are not considered significant.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): Not applicable (Milli-Q water)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 8N KOH(aq) (pre-diluted)
Duration of treatment / exposure:
Observations are made 3-minutes and 1-hour post-test item application
Duration of post-treatment incubation (if applicable):
The DMEM was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol over night at room temperature.
Number of replicates:
Duplicate; used for a 3-minute exposure to the test substance and two for a 1-hour exposure.
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 minute exposure
Run / experiment:
mean (n=2)
Value:
93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basis: mean. Time point: 3 minute exposure. Remarks: n=2 ; CV = 6.0 % ; Score in terms of percentage of negative control.
Irritation / corrosion parameter:
% tissue viability
Remarks:
1 hour exposure
Run / experiment:
mean (n=2)
Value:
121
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basis: mean. Time point: 1 hour exposure. Remarks: n=2 ; CV = 11 % ; Score in terms of percentage of negative control.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has previously demonstrated technical proficiency. Also see historic control data.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data are presented in the full study report. All values for the control groups were within the ranges of the current Historic Control Data. Historic Control Data: Negative Control OD570: 3-minutes (n=116): 1.258– 2.615 ; 1-hour (n=119): 1.371 – 2.371. Positive Control HCD data are presented within the full study report and the concurrent PC was within the specified ranges.

Table 1. Mean absorption and tissue viability in the in vitro skin corrosion test with the test item

Dose Group

Replicate

3 minute OD570 nm corrected (for blank)

Mean OD570 nm

Standard Deviation

3 minute

Mean viability (% of control)

CoV (%)

1 hour OD570 nm corrected (for blank)

Mean OD570 nm

Standard Deviation

1 hour

Mean viability (% of control)

CoV (%)

Blank (isopropanol)

 

0.0490

 

 

 

 

 

 

 

 

Negative Control

A

1.974

1.924

0.070

100

5.0

1.663

1.636

0.039

100

3.3

B

1.874

1.608

Positive Control

A

0.113

0.102

0.016

5.3

19.0

0.168

0.126

0.059

7.7

50.0

B

0.091

0.084

Test Item

A

1.735

1.791

0.078

93

6.0

1.867

1.983

0.165

121

11

B

1.846

2.100

CV : Coefficient of Variation between tissue replicates : CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

 

The positive control had a mean relative tissue viability of 7.7% after the 1-hour exposure. The absolute mean OD570 of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20-100% viability the Coefficient of Variation between tissue replicates was ≤ 11%, indicating that the test system functioned properly. All assay acceptance criteria were met.

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item is not considered to be corrosive to the skin.
Executive summary:

The study was performed to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test item in accordance with GLP using a human three dimensional epidermal model (EpiDerm (EPI-200)). The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 μL of the undiluted test item was added (with a pipette) into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μL Milli-Q water (negative control) and with 50 μL 8N KOH (positive control), respectively. The positive control had a mean relative tissue viability of 7.7% after the 1-hour exposure. The absolute mean OD570 of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20-100% viability the Coefficient of Variation between tissue replicates was ≤ 11%, indicating that the test system functioned properly.All assay acceptance criteria were met.Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 93% and 121%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive. Under the conditions of this study, the test item is not considered to be corrosive to the skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07-08-2019 to 23-09-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 67%) which does not affect the reliability of the study
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 67%) which does not affect the reliability of the study
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM Small Model
- Tissue batch number(s): Lot no.: 19-EKIN-38
- Production date: Not reported.
- Shipping date: Not reported. Although the CoA for the tissues QA is provided in the full study report.
- Delivery date: 17-09-2019 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: ca. 17-09-2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.0 °C, 5 ± 0.5% CO2 (actual: 36.3 – 37.1°C)
- Temperature of post-treatment incubation (if applicable): 37 ± 1.0 °C, 5 ± 0.5% CO2; for 42 hours. For further information see below.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. For the cell viability measurements: cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for approximately 72 hours. The amount of extracted formazan was determined spectrophotometrically.
- Observable damage in the tissue due to washing: Not applicable.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3.0 mg/mL MTT concentrated diluted (x10) to 0.3 mg/mL MTT solution prepared in assay medium
- Incubation time: For the cell viability measurements: cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for approximately 72 hours. The amount of extracted formazan was determined spectrophotometrically.
- Spectrophotometer: M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: Without reference filter
- Filter bandwidth: No reported.
- Linear OD range of spectrophotometer: Not reported.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Within NC and PC historic control ranges presented within the report.
- Barrier function: See above.
- Morphology: See above.
- Contamination: Not applicable.
- Reproducibility: The relative mean tissue viability for the positive control treated tissues was 5.7% relative to the negative control following the 15-minute exposure/42-hour incubation period. With standard deviation of 0.1%. The positive control acceptance criterion was therefore satisfied. The negative control triplicate treated tissues mean OD570 was 1.052 and the standard deviation of viability was 4.1%. The acceptance criterion was therefore satisfied. The test item triplicate treated tissues viability standard deviation was 2.4%. The mean OD570 for both the negative and positive controls were within the historical control ranges.
- Other: As an additional quality control step, the results of the assay are compared to the historical ranges for negative and positive controls generated by the testing laboratory to confirm the functioning of the test system.

NUMBER OF REPLICATE TISSUES: Three (3), triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: In the pretest for colour interference: the solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues. In the pre-test for direct MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT. It was unnecessary to run killed tissues.
- Procedure used to prepare the killed tissues (if applicable): Not applicable.
- N. of replicates : Not applicable.
- Method of calculation used: Not applicable.
- Other: In the assessment of Colour Interference with the MTT endpoint, the solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause colour interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One (n=1)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if e.g. the viability after 15-minutes exposure / 42-hours incubation is less than or equal to 50%,.
- The test substance is considered to be non-irritating to skin if e.g. the viability after 15-minutes exposure / 42-hours incubation is greater than 50%.
- Cut off points in accordance with OECD TG 439 and the GHS and CLP Classification systems.
- Skin irritation is expressed as the remaining cell viability after exposure to the test substance at exposure times 15-minutes / 42-hours incubation. Where necessary, direct MTT reduction, colour interference modifications were completed.

OTHER:
EpiSkin RHE Small Model (Lot no.: 19-EKIN-038). The test consists of topical application of the test item -one on the skin tissue for 15 minutes. After exposure the skin tissue is thoroughly rinsed to remove the test item and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect is performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. The EPISKIN Small Model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 23 hours at 37°C. Maintenance medium and Assay medium were supplied by a recognised supplier (documented in the full study report).

Application of test item and rinsing:
Test item: Tissues were treated with 10 µL test item for 15 minutes exposure period. Negative control: 10 µL PBS (negative control) was similarly applied. Positive control: 10 µLl SDS (5% w/v) was respread after 7 minutes contact time to maintain distribution of the SDS - for the remainder of the contact period. This was not deemed necessary in the negative control or test item definitive test group. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 67 – 95%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.1°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on testing laboratory historical data these deviations are not considered significant.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): Not applicable. (Phosphate buffered saline, pre-diluted)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% SDS (pre-diluted)
Duration of treatment / exposure:
15 ± 0.5 minutes at room temperature, after which the tissues were washed with phosphate buffered saline to remove residual test item.
Duration of post-treatment incubation (if applicable):
Subsequently the skin tissues were incubated for 42 hours at 37°C.
Number of replicates:
Triplicate; treatment and concurrent negative control and positive control groups
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean (n=3)
Value:
9.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basis: mean. Time point: 15 minute exposure. Remarks: n=3; SD = 2.4% ; Score in terms of percentage of negative control.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has previously demonstrated technical proficiency. Also see historic control data.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data are presented in the full study report. All values for the control groups were within the ranges of the current Historic Control Data. Historic Control Data (n=173): the mean OD of the positive control was 0.12; range 0.023 to 0.449. In this same period the mean OD of the negative control was 0.96; range 0.422 – 1.547.

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test item

Item

OD570 of tissues

Mean OD570 of triplicate tissues

±SD of OD570

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

1.027

1.052

0.043

100*

4.1

1.028

1.101

Positive Control Item

0.061

0.060

0.001

5.7

0.1

0.060

0.059

Test Item

0.129

0.099

0.026

9.4

2.4

0.084

0.085

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%.

Values are corrected for background absorption (0.469). Isopropanol was used to measure background adsorption.

Negative control: Phosphate buffered saline (PBS)

Positive control: 5% (aq.) Sodium dodecyl sulphate (SDS)

 

The relative mean tissue viability for the positive control treated tissues was 5.7% relative to the negative control treated tissues and the standard deviation value of the viability was 0.1%. The mean OD570 for the negative control treated tissues was 1.052 and the standard deviation value of the viability was 4.1%. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 2.4%. All assay acceptance criteria were met.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item is considered to be irritating to the skin.
Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test item together with negative and positive controls. 10 μL of the undiluted test item was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μL PBS (negative control) and 3 tissues with 10 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 5.7% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 9.4%. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was below 50% after 15 minutes treatment it is considered to be irritant. Under the conditions of this study the test item is irritant to the skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
key study
Study period:
06-02-2020 to 14-04-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: May 2018 ; signature: August 2018
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Recognised supplier (documented in the full study report)
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): ca. 7 weeks old (typically).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated heads from ca. 7 week old donors were collected from the abattoir at transported at ambient. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at the test facility and processed within 2 hours of collection by the test facility. After removing the individual head from the plastic box, it was put on soft paper. The eyelids were carefully removed, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit (by standardised procedure), then the eye was placed onto damp paper and the nictitating membrane was removed with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained. Eyes where then clamped in the superfusion apparatus and then re-examined to ensure good condition. Then the eyes were acclimatised for 45 to 60 minutes at (32±1.5°C)
- Time interval prior to initiating testing: < 24 hours. Eyes were prepared for testing immediately on same day arrival within 2 hours of collection.
- indication of any existing defects or lesions in ocular tissue samples: None. Only eyes free from damage
- Indication of any antibiotics used: None reported.
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): 0.03 mL (or 30 µL of the test item)
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.
Duration of treatment / exposure:
The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of physiological saline.
Controls (negative and positive control items) were similarly applied to the cornea in the negative and positive control groups respectively.
Observation period (in vivo):
Treated eyes were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been rinsed with the physiological saline.
Number of animals or in vitro replicates:
Triplicate (n=3)
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES: Full details are provided in the full study report. Freshly isolated heads from ca. 7 week old donors were collected from the abattoir at transported at ambient. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at the test facility and processed within 2 hours of collection by the test facility. After removing the individual head from the plastic box, it was put on soft paper. The eyelids were carefully removed, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit (by standardised procedure), then the eye was placed onto damp paper and the nictitating membrane was removed with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained. Eyes where then clamped in the superfusion apparatus and then re-examined to ensure good condition. Then the eyes were acclimatised for 45 to 60 minutes at (32±1.5°C)

EQUILIBRATION AND BASELINE RECORDINGS: Taken at zero time after 45 to 60 minutes incubation prior to exposure.

NUMBER OF REPLICATES: Triplicate (3) for test item, positive control and negative controls.

NEGATIVE CONTROL USED: Yes,.

SELECTION AND PREPARATION OF ISOLATED EYES: Full details are provided in the full study report. Freshly isolated heads from ca. 7 week old donors were collected from the abattoir at transported at ambient. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at the test facility and processed within 2 hours of collection by the test facility. After removing the individual head from the plastic box, it was put on soft paper. The eyelids were carefully removed, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit (by standardised procedure), then the eye was placed onto damp paper and the nictitating membrane was removed with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained. Eyes where then clamped in the superfusion apparatus and then re-examined to ensure good condition. Then the eyes were acclimatised for 45 to 60 minutes at (32±1.5°C)

EQUILIBRATION AND BASELINE RECORDINGS: Taken at zero time after 45 to 60 minutes incubation prior to exposure.

NUMBER OF REPLICATES: Triplicate (3) for test item, positive control and negative controls.

NEGATIVE CONTROL USED: Yes,.

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED : Benzalkonium chloride (5%)

APPLICATION DOSE AND EXPOSURE TIME: Application dose: 0.03 mL (or 30 µL of the test item) ; Exposure time: 10 seconds.

OBSERVATION PERIOD: prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after decontaminated with saline.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Test item remained in place for 10 seconds and then rinsed with 20 mL physiological saline. Treated eyes were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes after the eyes had been rinsed with the physiological saline.
- Indicate any deviation from test procedure in the Guideline: Not applicable.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Microscope.
- Damage to epithelium based on fluorescein retention: Microscope.
- Swelling: measured with optical pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: Yes.
- Others (e.g, histopathology): Not applicable.

SCORING SYSTEM:
- Mean corneal swelling (%): See tables 1 to 2.
- Mean maximum opacity score: See tables 1 to 2.
- Mean fluorescein retention score at 30 minutes post-treatment: See tables 1 to 2.

DECISION CRITERIA: In accordance with guideline OECD TG 438.
Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium, sticking of test item to the cornea). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test item exposure) were determined at each of the above time points.
ICE classes were determined based on a predetermined range in accordance with the criteria given in OECD TG 438.
The overall in vitro irritancy classification of the test item was determined by using all the classification information and criteria given in OECD TG 438 and the UN GHS classification referenced in the guideline. Full details are provided in the full study report.

A test was considered acceptable if the concurrent negative or vehicle/solvent items and the concurrent positive controls were within the historic control data range and/or identified as GHS Non Classified and GHS Category 1, respectively
Irritation parameter:
cornea opacity score
Run / experiment:
mean (n=3)
Value:
0.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
mean (n=3)
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
percent corneal swelling
Run / experiment:
mean (n=3)
Value:
-3.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: value %; ICE Class I
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: Not applicable. Applicant assessment of the data indicates that the positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

Table 1. Ocular reactions

Test Item

 

 

Maximal mean score for corneal opacity

0.33

ICE Class I

Mean score of Fluorescein Retention

0.00

ICE Class I

Corneal swelling

-3.9%

ICE Class I

 

 

 

Positive Control Item

 

 

Maximal mean score for corneal opacity

4.00

ICE Class IV

Mean score of Fluorescein Retention

2.83

ICE Class IV

Corneal swelling

23.1%

ICE Class III

 

 

 

Negative Control Item

 

 

Maximal mean score for corneal opacity

0.00

ICE Class I

Mean score of Fluorescein Retention

0.00

ICE Class I

Corneal swelling

0.00%

ICE Class I

 

- Corneal Opacity Scores

Maximum score was 0.5 (n=2 of 3) in the test item group. Maximum score was 0.0 in the negative control. In the positive control group the Maximum score was 4.0. No morphological effects were noted in the test item or negative control item treated eyes. In the positive control group, severe loosening of epithelium was observed in one eye at 75 minutes after the post-treatment rinse.

 

- Fluorescein Retention Scores

Maximum score was 0.0 (n=3 of 3) in the test item group and negative control. In the positive control group the Maximum score was 3.00.

 

Table 2. Individual scoring - test item

End Point

Eye Number

Time (after decontamination)

0 minutes

30 minutes

75 minutes

120 minutes

180 minutes

240 minutes

Corneal Opacity

12

0

0

0

0

0

0

13

0

0

0

0.5

0.5

0.5

14

0

0

0

0.5

0.5

0.5

Mean

0.00

0.00

0.00

0.33

0.33

0.33

ICE Class

I

Fluorescein Retention

12

0.5

0.5

 

 

 

 

13

0.0

0.0

 

 

 

 

14

0.0

0.0

 

 

 

 

Mean difference (30 - 0 min)

 

0.00

 

 

 

 

ICE Class

I

Corneal Thickness

3A

0.60

0.60

0.60

0.58

0.58

0.58

6A

0.62

0.62

0.62

0.62

0.60

0.60

8A

0.60

0.60

0.60

0.59

0.57

0.57

Mean

0.61

0.61

0.61

0.60

0.58

0.58

Mean Corneal Swelling (%)

 

0.0

0.0

-1.7

-3.9

-3.9

ICE Class

I

ICE Classes Combined:

3 x I

Classification:

Category 1

 

The test was considered acceptable since the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non Classified and GHS Category 1, respectively.

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item was considered to be non-irritant.
Executive summary:

The study was performed according to OECD TG 438 in accordance with GLP to assess the eye irritancy potential of the test item in isolated chicken eyes. The method involves evaluation of the eye hazard potential of a test chemical as measured by its ability to induce toxicity in an enucleated chicken eye. Toxic effects to the cornea are measured by (i) a qualitative assessment of opacity (ii) a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention) (iii) a quantitative measurement of increased thickness (swelling) and (iv) a qualitative evaluation of macroscopic morphological damage to the surface. The method is also able to identify substances not requiring UN GHS classification.After the zero reference measurements, the eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds exposure time, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 μL test item. The three positive control eyes were treated in a similar way with 30 μL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated. For the test item the mean corneal opacity was 0.33 (ICE Class I). The mean fluorescein retention was 0.0 (ICE Class I) and the mean corneal thickness across 30, 75, 120, 180 and 240 minutes was ICE Class V with maximal corneal swelling -3.9%. No other morphological effect was observed. The negative control gave a prediction of GHS non-classified for eye irritation (ICE Class I) across all categories. The positive control gave a prediction of GHS Category 1 (ICE Class IV across cornel opacity and fluorescein retention categories and Class III in mean corneal thickness) signifying that the test system performed adequately. Under the conditions of this study, the test item was considered to be non-irritant.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-08-2019 to 16-10-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
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:
MatTek Corporation EpiOcular™ Eye Irritation Test (OCL-200-EIT)
Deviations:
no
GLP compliance:
yes
Species:
other: human-derived epidermal keratinocyte tissues
Strain:
not specified
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: The test method is indicated as a validated test method recommended by OECD and/or recognised internationally for the sequential testing strategy for the assessment of ocular irritation.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27433 Kit A, Certificate of Analysis provided in the full study report).
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 of the test item
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.
Duration of treatment / exposure:
30 ± 2 minutes at 37.0 ± 1.0°C
Duration of post- treatment incubation (in vitro):
120 ± 15 minutes at 37°C post treatment / rinsing incubation
- Rinsing consisted of Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a prelabelled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post- Soak).
- Dried culture inserts were then transferred to 6-well plates containing 1.0 mL of warm Assay Medium and were post-treatment incubated for 120 ± 15 minutes at 37°C.
Number of animals or in vitro replicates:
Two tissues were used for each treatment group (test item, negative control and positive control).
Details on study design:
- Details of the test procedure used: See below.
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27493 Kit C, Certificate of Analysis provided in the full study report).
- Doses of test chemical and control substances used: 50 µL of the test item / 50 µL Milli-Q water (negative control) / 50 µL methyl acetate (positive control)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Replicate tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37°C in 1.0 mL fresh pre-warmed supplemented DMEM Assay Medium. All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 63 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.1°C). Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door were considered not to impact the study based on historic laboratory data. Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. The liquid test item was applied undiluted (50 μL) directly on top of two tissues. Test item was spread to match the size of the tissues. Two tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues with 50 µL Methyl Acetate (positive control) respectively. After the exposure period with test item (30 ± 2 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 120 ± 15 minutes at 37°C.
- Justification for the use of a different negative control than ultrapure H2O (if applicable): Not applicable.
- Justification for the use of a different positive control than neat methyl acetate (if applicable): Not applicable.
- Description of any modifications to the test procedure: Not applicable.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable):
Colour-interference: was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the tissues during the exposure. To assess the colour interference, 50 μL of test item or 50 μL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark. Furthermore, 50 μL of test item or 50 μL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking. The absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with an M200 Pro Plate Reader. Centrifugation was not considered necessary. If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement. Conclusion: addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of -0.0010 and 0.0016, respectively. Therefore it was concluded that the test item did not induce colour interference.
Direct reduction of MTT: The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 μL of test item was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark. A negative control, 50 μL sterile Milli-Q water was tested concurrently. If the MTT solution colour turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay. Conclusion: the test item did not directly interact with MTT and therefore was a direct MTT reducer.
As a result: NSMTT, NSCliving and NSCkilled were not required.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): Duplicate.
- 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: TECAN Infinite® M200 Pro Plate – spectrophotometrically.
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): Not applicable.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: According to the OECD TG 492 and GHS system.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range: 1.077– 2.090 and within the OECD TG 492, acceptability range of OD > 0.8 and < 2.5 and/or the difference in viability was < 10% between replicates. The Positive Control gave a response within the historic control range: 2.11 – 48.25%. The current HCD was provided in the full study report.
- Complete supporting information for the specific RhCE tissue construct used: Attached to the full study report.
- Reference to historical data of the RhCE tissue construct: Full historic control data is attached to the full study report.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The test assay is validated at the test laboratory and this information is in the public domain.
- Positive and negative control means and acceptance ranges based on historical data: See above.
- Acceptable variability between tissue replicates for positive and negative controls: < 10 %.
- Acceptable variability between tissue replicates for the test chemical: Yes.
Irritation parameter:
other: Mean tissue viability (% of negative control)
Remarks:
n=2
Run / experiment:
mean (n=2) - test item
Value:
56
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Mean tissue viability (% of negative control)
Remarks:
n=2
Run / experiment:
mean (n=2) - positive control
Value:
21
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test assay is validated at the test laboratory and this information is in the public domain.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: See 'details on study design' for further information. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range: 1.077– 2.090 and within the OECD TG 492, acceptability range of OD > 0.8 and < 2.5 and/or the difference in viability was < 10% between replicates. The Positive Control gave a response within the historic control range: 2.11 – 48.25%. The current HCD was provided in the full study report.

Table 1.0 : Mean absorption and Mean Tissue Viability in the EPIOCULAR Test

 

A

(OD570)

B

(OD570)

Mean

(OD570)

SD

Mean tissue viability (percentage of control)

Difference between two tissues
(percentage)

Negative control

1.799

1.731

1.765

± 0.048

100

3.9

Test Item

0.946

1.031

0.988

±0.060

56

4.8

Positive control

0.453

0.292

0.373

± 0.113

21

9.1

 

Acceptability of the Assay

a) The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.

b) The mean relative tissue viability of the positive control should be < 50% relative to the negative control.

c) The difference between the % tissue viabilities of the two identically treated replicates should be < 20%.

d) The non-specific colour of the test item should be < 50% relative to the negative control OD.

All assay acceptability criteria were met.

Interpretation of results:
other: no prediction can be made
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, since the test item relative mean viability was < 60% the test item is considered potentially eye irritant or corrosive. However, under the GHS criteria no prediction can be made about the categorization of the test item.
Executive summary:

The study was performed to assess the eye irritancy potential of the test item using the EpiOcular EIT model under OECD TG 492 guideline in accordance with GLP. The test item was topically applied for 30 ± 2 minutes at 37.0 ± 1.0°C. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. The tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. In pre-testing the test item did not directly interact with MTT and therefore was a direct MTT reducer. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test item compared to the negative control tissues was 56%. The positive control had a mean cell viability of 21% after 30 ± 2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptability criteria. The difference between the percentages of viability of two tissues treated identically was less than 10%, indicating that the test system functioned properly. Under the conditions of this study, since the test item relative mean viability was < 60% the test item is considered potentially eye irritant or corrosive. However, under the GHS criteria no prediction can be made about the categorization of the test item under the experimental conditions described.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Corrosion:

Key study: in vitro, OECD TG 431, 2019 : The study was performed to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test item in accordance with GLP using a human three dimensional epidermal model (EpiDerm (EPI-200)). The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 μL of the undiluted test item was added (with a pipette) into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μL Milli-Q water (negative control) and with 50 μL 8N KOH (positive control), respectively. The positive control had a mean relative tissue viability of 7.7% after the 1-hour exposure. The absolute mean OD570 of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20-100% viability the Coefficient of Variation between tissue replicates was ≤ 11%, indicating that the test system functioned properly.All assay acceptance criteria were met.Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 93% and 121%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive. Under the conditions of this study, the test item is not considered to be corrosive to the skin.

 

Skin Irritation:

Key study : in vitro, OECD TG 439, 2020 : The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test item together with negative and positive controls. 10 μL of the undiluted test item was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μL PBS (negative control) and 3 tissues with 10 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 5.7% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 9.4%. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was below 50% after 15 minutes treatment it is considered to be irritant. Under the conditions of this study the test item is irritant to the skin.

 

Eye Irritation:

Key study : in vitro, OECD TG 438, 2020 : The study was performed according to OECD TG 438 in accordance with GLP to assess the eye irritancy potential of the test item in isolated chicken eyes. The method involves evaluation of the eye hazard potential of a test chemical as measured by its ability to induce toxicity in an enucleated chicken eye. Toxic effects to the cornea are measured by (i) a qualitative assessment of opacity (ii) a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention) (iii) a quantitative measurement of increased thickness (swelling) and (iv) a qualitative evaluation of macroscopic morphological damage to the surface. The method is also able to identify substances not requiring UN GHS classification.After the zero reference measurements, the eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds exposure time, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 μL test item. The three positive control eyes were treated in a similar way with 30 μL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated. For the test item the mean corneal opacity was 0.33 (ICE Class I). The mean fluorescein retention was 0.0 (ICE Class I) and the mean corneal thickness across 30, 75, 120, 180 and 240 minutes was ICE Class V with maximal corneal swelling -3.9%. No other morphological effect was observed. The negative control gave a prediction of GHS non-classified for eye irritation (ICE Class I) across all categories. The positive control gave a prediction of GHS Category 1 (ICE Class IV across cornel opacity and fluorescein retention categories and Class III in mean corneal thickness) signifying that the test system performed adequately. Under the conditions of this study, the test item was considered to be non-irritant.

 

Key study : in vitro, OECD TG 492, 2019 : The study was performed to assess the eye irritancy potential of the test item using the EpiOcular EIT model under OECD TG 492 guideline in accordance with GLP. The test item was topically applied for 30 ± 2 minutes at 37.0 ± 1.0°C. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. The tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. In pre-testing the test item did not directly interact with MTT and therefore was a direct MTT reducer. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test item compared to the negative control tissues was 56%. The positive control had a mean cell viability of 21% after 30 ± 2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptability criteria. The difference between the percentages of viability of two tissues treated identically was less than 10%, indicating that the test system functioned properly. Under the conditions of this study, since the test item relative mean viability was < 60% the test item is considered potentially eye irritant or corrosive. However, under the GHS criteria no prediction can be made about the categorization of the test item under the experimental conditions described.

 

Respiratory Irritation:

No data available.

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for skin irritation: category 2: H315: causes skin irritation

 

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation