Registration Dossier

Administrative data

Description of key information

Skin corrosion in vitro: The test item was determined to be non-corrosive to skin becausemean tissue viability of test item-treated tissues compared to the negative control was 95 % after an exposure period of 3 minutes and 95 % after and exposure period of 60 minutes(OECD 431).

 

Skin irritation in vitro: The test item was determined to be non-irritant to skin because mean tissue viability relative to the negative control was 92 % after an exposure period of 15 minutes (OECD 439).

 

Eye damage/irritation in vitro: The test item was determined not to cause eye damage/irritation because the mean in vitro irritancy score was 1.4 after an exposure period of 10 minutes (OECD 437).

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:
key study
Study period:
08 January 2018 to 12 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
other: human
Cell type:
non-transformed keratinocytes
Cell source:
other: accredited institutions after consent obtained from donor or donor's next of kin
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
INTRODUCTION
- The objective of this study was to evaluate the test item for its ability to induce skin corrosion.
- For this purpose, test material was topically applied on a human three dimensional epidermal model.

TEST ITEM CHARACTERISATION
- The study sponsor provided information on identity, purity, composition and stability of the test item.

RESERVE SAMPLES
- For each batch (lot) of test item, a reserve sample was collected and maintained under the appropriate storage conditions by the Test Facility and destroyed after the expiration date.

TEST ITEM INVENTORY AND DISPOSITION
- Records of the receipt, distribution, and storage of test item(s) were maintained.
- With the exception of reserve samples, all unused Sponsor-supplied test item were discarded or returned to the Sponsor after completion of the scheduled program of work.
- Records of the decisions made were kept at the Test Facility.

TEST SYSTEM
- The test is based on the experience that corrosive chemicals show cytotoxic effects following short-term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin corrosion potential of a test item by assessment of its effect on a three dimensional human epidermis model.
- The test consists of topical application of test material on the skin tissue for 3-minutes 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.
- EpiDerm Skin Model (EPI-200, Lot no. 27667, kit H and G, see Appendix 4, attached).
- The 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.

TEST FOR INTERFERENCE OF THE TEST ITEM WITH THE MTT ENDPOINT
- A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly
reduce MTT.
- The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

TEST FOR COLOUR INTERFERENCE BY THE TEST ITEM
- The test material 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 skin tissues during the 1-hour exposure.
- To assess the colour interference, 50 μL of the test item or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and checked to establish whether a blue / purple colour change was observed.

TEST FOR REDUCTION OF MTT BY THE TEST ITEM
- The test material 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 the test item or 50 μL Milli-Q
water as a negative control were added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C. At the end of the exposure time the mixture was checked to establish whether a blue / purple colour change or a blue / purple precipitate was observed.

TEST SYSTEM SET UP
- A diagram of the application procedure is shown in Figure 1 (attached).
- 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.
- Supplemented DMEM (Dulbecco’s Modified Eagle’s Medium) serum free was supplied by MatTek Corporation.
- MTT concentrate (5 mg/mL; MatTek Corporation) was diluted (1:5) with MTT diluent (supplemented DMEM; MatTek Corporation).
- 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 to 100 % (actual range 47 to 84 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.0 - 37.3 °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 laboratory historical data these deviations are considered not to affect the study integrity.

TEST ITEM PREPARATION
- No correction was made for the purity/composition of the test item.
- The liquid test item was applied undiluted (50 μL) directly on top of the tissue.

APPLICATION / TREATMENT OF THE TEST ITEM
- The skin tissues were kept in the refrigerator from the day they were received.
- The next day, at least 1 hour before the assay was started, the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue (see figure 1, attached).
- The plates were incubated for approximately 2.5 hours at 37.0 ± 1.0 °C.
- The medium was replaced with fresh DMEM just before the test item was applied.
- 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.
- Undiluted test item (50 µL) was added into the 6-well plates on top of the skin tissues.
- For the negative and positive controls, 2 tissues were treated with 50 μL Milli-Q water
(negative control) and 2 tissues were treated with 50 μL 8N KOH (positive control) for both
the 3-minute and 1-hour time point.
- After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) 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.

CELL VIABILITY MEASUREMENT
- 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 (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with a TECAN Infinite M200 Pro Plate Reader.

ACCEPTANCE CRITERIA
- The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be < 15 %.
c) In the range 20 to 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
- All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
- A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50 %.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with
the test item is decreased below 15 %.
- A test item is considered non-corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50 %.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15 %.

CALCULATION OF CELL VIABILITY
- Optical Density readings were transferred into Microsoft Excel to allow further calculations
to be performed.
- The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw) using the equation ODc = ODraw – ODbl
- The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT).
- The %Viability for each sample and positive control is calculated using the equation %Viability = (ODc/mean ODlt_u+MTT) * 100.

COMPUTERISED SYSTEMS
- Critical computerized systems used in the study are listed below:
a) REES Centron (version SQL 2.0) for temperature and humidity (laboratory facilities data collection).
b) Magellan Tracker (version 7.0) for optical density measurement.
- All computerised systems used in the conduct of the study have been validated. When a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 μL
Duration of treatment / exposure:
3 minutes and 1 hour
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
Two
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
95
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60-minute exposure
Value:
95
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
RESULTS
- The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed, it was concluded that the test item did not interfere with the MTT endpoint.
- The mean absorption at 570 nm measured after treatment with test material and controls are presented in Table 1 (attached). The individual OD570 measurements are presented in Appendix 2 (attached).
- Mean tissue viability obtained after 3-minute and 1-hour treatments with test material compared to the negative control tissues is shown in Table 2 (attached).
- Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained both after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 95 %. Because the mean relative tissue viability for the test material was not below 50 % after 3 minutes treatment and not below 15 % after 1-hour treatment, the substance is considered to be not corrosive.
- The absolute mean OD570 (optical density at 570 nm) 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 (See Appendix 3, attached).
- The mean relative tissue viability following the 1-hour exposure to the positive control was 4.7%. In the range of 20 to 100% viability the Coefficient of Variation between tissue replicates was ≤ 14%, indicating that the test system functioned properly (see Table 3, attached).
Interpretation of results:
GHS criteria not met
Conclusions:
The test item was determined to be not corrosive in the in vitro skin corrosion test under the experimental conditions described.
Executive summary:

GUIDELINE

The test item was evaluated for its ability to induce skin corrosion on a human three-dimensional epidermal model (EpiDerm (EPI-200)) in accordance with OECD Guideline 431 “In Vitro Skin Corrosion: reconstructed human epidermis (RHE) test method” (adopted 29 July 2016) and EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test" Official Journal of the European Union No. L142, 31 May 2008.

 

METHODS

The supplied batch of test material was a clear colourless liquid. The test item was applied undiluted (50 μL) directly on top of the skin tissue. The positive control had a mean relative tissue viability of 4.7% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) 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 to 100 % viability the Coefficient of Variation between tissue replicates was ≤ 14 %, indicating that the test system functioned properly. Skin corrosion was expressed as the remaining cell viability after exposure to the test item.

 

RESULTS

The relative mean tissue viability obtained both after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 95%. Because the mean relative tissue

viability for the test material was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, the substance was considered to be non-corrosive.

 

CONCLUSION

The test item was determined to be not corrosive in the in vitro skin corrosion test under the experimental conditions described.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 March 2018 to 12 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Source strain:
other: adult
Vehicle:
unchanged (no vehicle)
Details on test system:
BACKGROUND
- The test is based on the experience that irritant chemicals show cytotoxic effects following short term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin irritation potential of a test item by assessment of its effect on a three dimensional human epidermis model.
- The test consists of topical application of the test item 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.

TEST ITEM CHARACTERISATION
- The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item(s).
- A Certificate of Analysis or equivalent document was provided to the Test Facility.

RESERVE SAMPLES
- For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility and destroyed after the expiration date.

TEST SYSTEM
- EPISKIN-SM, 0.38 cm2, Batch number: 18-EKIN-010 (SkinEthic Laboratories, Lyon, France).
- This 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.

TEST FOR INTERFERENCE OF THE TEST ITEM WITH THE MTT ENDPOINT
- A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.
- The test material was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model. Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.

TEST SYSTEM SET-UP
- On the day of receipt, tissues were transferred to 12-well plates and pre-incubated with pre warmed Maintenance Medium for 22 hours at 37°C (see Figure 1, attached).
- Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

MTT MEDIUM
- MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/mL in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL).

ENVIRONMENTAL CONDITIONS
- 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 to 100% (actual range 64 - 85%), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 to 37.4 °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 laboratory historical data these deviations are considered not to affect the study integrity.

TEST ITEM PREPARATION
- No correction was made for the purity/composition of the test item.
- The liquid test item was applied undiluted (25 μL) directly on top of the tissue.

APPLICATION / TREATMENT OF THE TEST ITEM
- The test was performed on a total of 3 tissues per test item together with negative and positive controls.
- Undiluted test item (25 μL) was added into 12-well plates on top of the skin tissues.
- Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 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.

CELL VIABILITY MEASUREMENT
- After incubation, 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 (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 69 hours.
- The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
- The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤ 18.
b) The mean relative tissue viability of the positive control should be ≤ 40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤ 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤ 18.
- All results presented in the tables of the report were calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
- A test item is considered irritant in the skin irritation test if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

CALCULATION OF CELL VIABILITY
- Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
- The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw) using the equation ODc = ODraw - ODbl
- The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
- The %Viability for each sample and positive control was calculated using the equation %Viability = (ODc/mean ODlt_u+MTT) * 100

CRITICAL COMPUTERISED SYSTEMS
- Temperature and humidity (laboratory facilities) Data collection: REES Centron (version SQL 2.0)
- Optical Density Measurement: Magellan Tracker (version 7.0)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 μL
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test
Value:
92
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- The test item was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model. Because no colour changes were observed it was concluded that the test item did not interact with the MTT endpoint.
- The mean absorption at 570 nm measured after treatment with the test item and controls are presented in Table 1 (attached).
- The individual OD570 measurements are presented in Appendix 2 (attached).
- Table 2 (attached) shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with test item compared to the negative control tissues was 92%. Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.
- The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 3.3%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (see Appendix 3, attached). The standard deviation value of the percentage viability of three tissues treated identically was ≤ 11%, indicating that the test system functioned properly.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonised System of Classification and Labelling of Chemicals (GHS) of the United Nations.
Executive summary:

GUIDELINE

The objective of the study was to evaluate the test item for its ability to induce skin irritation

on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM)). The investigation was conducted in accordance with OECD Guideline 439.In vitro Skin Irritation: Reconstructed Human Epidermis Test Method,(adopted 28 July 2015) and EC Guideline No. 440/2008.Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In vitro Skin Irritation: Reconstructed Human Epidermis Model Test ".Official Journal of the European Union No. L142; Amended byEC No. 640/2012 OJ No. L193, 20 July 2012.

 

METHODS

The test item was a clear colourless liquid. The test item was applied undiluted (25 μL) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42-hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation was expressed as the remaining cell viability after exposure to the test item.

 

RESULTS

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 92%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant. The positive control had a mean cell viability of 3.3% after 15 ± 0.5 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 standard deviation value of the percentage Viability of three tissues treated identically was≤11%, indicating that the test system functioned properly.

 

CONCLUSION

The test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonised System of Classification and Labelling of Chemicals (GHS) of the United Nations.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes
Species:
cattle
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 μL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 ± 10 minutes
Number of animals or in vitro replicates:
Three
Details on study design:
TEST SYSTEM
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

PREPARATION OF CORNEAS
- The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1 % (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.

CORNEA SELECTION AND OPACITY READING
- After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany).
- The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
- The medium from the anterior compartment was removed and 750 µl of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1 °C.
- After incubation, the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1 °C.
- After completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

OPACITY MEASUREMENT
- The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- The opacity value (measured with the device OP-KIT) was calculated using the equation Opacity = [(I0/I) – 0.9894] / 0.0251 where I0 = the empirically determined illuminance through a cornea holder but with windows and medium; I = the measured illuminance through a holder with cornea.
- The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading.
- The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
- The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

APPLICATION OF SODIUM FLUORESCEIN
- Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
- The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1 °C.

PERMEABILITY DETERMINATIONS
- After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite M200 Pro Plate Reader).
- Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
- The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

ACCEPTABILITY CRITERIA
- The assay is considered acceptable if:
(a) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
(b) The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
- All results presented in the tables of the report were calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score using the equation In vitro irritancy score (IVIS) = mean opacity value + (15 * mean OD490 value).
- Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

COMPUTERISED SYSTEMS
- Optical Density Measurement: Magellan Tracker version 7.0
- Temperature (laboratory facilities) data collection: REES Centron version SQL 2.0
Irritation parameter:
in vitro irritation score
Remarks:
mean
Value:
1.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
RESULTS
- Table 1 (attached) summarises the opacity, permeability and in vitro irritancy scores of the test item and the controls.
- The opacity, permeability and in vitro scores of the individual corneas are shown in Table 2 to 5 (attached).
- The individual in vitro irritancy scores for the negative controls ranged from -0.1 to 0.1.
- The individual positive control in vitro irritancy scores ranged from 48 to 88 (see Table 5, attached). The corneas treated with the positive control item were turbid after the 10 minutes
of treatment.
- The corneas treated with test item showed opacity values ranging from 0.6 to 1.7 and permeability values ranging from 0.015 to 0.032. The corneas were clear after the 10 minutes
of treatment.
- No pH effect of the test item was observed on the rinsing medium.
- The in vitro irritancy scores ranged from 1.1 to 2.1 resulting in a mean in vitro irritancy score of 1.4 after 10 minutes of treatment with the test material.

DISCUSSION

- The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 64 and within two standard deviations of the current historical positive control mean (Appendix 3, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

- The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.4 after 10 minutes of treatment.

Interpretation of results:
GHS criteria not met
Conclusions:
Since the test iteminduced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

GUIDELINE

The objective of this study was to evaluate the eye hazard potential of the test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The investigation was conducted in accordance with 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, (adopted July 26, 2013).

 

METHODS

This report describes the potency of chemicals to induce serious eye damage using isolated

bovine corneas. Eye damage was tested through topical application for 10 minutes. The test item was a clear colourless liquid. The test item was applied as it is (750 μL) directly on top of the corneas. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.

 

RESULTS

The mean in vitro irritancy score of the positive control (Ethanol) was 64 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro

irritancy score of 1.4 after 10 minutes of treatment.

 

CONCLUSION

Since the test iteminduced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion in vitro

The test item was evaluated for its ability to induce skin corrosion on a human three-dimensional epidermal model (EpiDerm (EPI-200)) in accordance with OECD Guideline 431 “In Vitro SkinCorrosion: reconstructed human epidermis (RHE) test method” (adopted 29 July 2016) and ECGuideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test" Official Journal of the EuropeanUnion No. L142, 31 May 2008.

The supplied batch of test material was a clear colourless liquid. The test item was appliedundiluted (50 μL) directly on top of the skin tissue. The positive control had a mean relative tissue viability of 4.7% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of thenegative control tissues waswithin the acceptance limits of OECD 431 (lower acceptance limit0.8 and upper acceptancelimit2.8) and the laboratory historical control data range. In the range of 20to100% viability the Coefficient of Variation between tissue replicates was14%, indicating that the test system functioned properly.Skin corrosion was expressed as the remaining cell viability after exposure to the test item.

 

The relative mean tissue viability obtained both after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 95 %. Because the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment, the substance was considered to be non-corrosive.

Skin irritation in vitro

The objective of the study was to evaluate the test item for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM)). The investigation was conducted in accordance with OECD Guideline 439.In vitro Skin Irritation: Reconstructed Human Epidermis Test Method,(adopted 28 July 2015) and EC Guideline No. 440/2008.Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In vitro Skin Irritation: Reconstructed Human Epidermis Model Test ".Official Journal of the European Union No. L142; Amended byEC No. 640/2012 OJ No. L193, 20 July 2012.

 

The test item was a clear colourless liquid. The test item was applied undiluted (25 μL) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42-hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction ofmitochondrial dehydrogenase activity measured by formazan production from MTT at the end of thetreatment. Skin irritation was expressed as the remaining cell viability after exposure to the test item.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test itemcompared to the negative control tissues was 92%. Since the mean relative tissue viability forthe test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to benon-irritant. The positive control had a mean cell viability of 3.3% after 15 ± 0.5 minutes exposure. Theabsolute mean OD570 (optical density at 570 nm) of the negative control tissues was within thelaboratory historical control data range. The standard deviation value of the percentage Viability ofthree tissues treated identically was11%, indicating that the test system functioned properly.

 

The test item is non-irritant in the in vitro skin irritation test under theexperimental conditionsdescribed in this report and should not be classified according to theGlobally Harmonised System ofClassification and Labelling of Chemicals (GHS) of theUnited Nations.

Eye damage/irritation in vitro

The objective of the study was to evaluate the eye hazard potential of the test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The investigation was conducted in accordance with 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, (adopted July 26, 2013).

 

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. Eye damage was tested through topical application for 10 minutes. The test item was a clear colourless liquid. The test item was applied as it is (750 μL) directly on top of the corneas. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.

 

The mean in vitro irritancy score of the positive control (Ethanol) was 64 and was within twostandard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.4 after 10 minutes of treatment.

Since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Justification for classification or non-classification

Skin corrosion in vitro: Mean tissue viability of test item-treated tissues compared to the negative control was determined to be50 % after 3-minute exposure and15 % after 60-minute exposure. Classification for skin corrosivity in accordance with Regulation (EC) No1272/2008 is therefore not required.

 

Skin irritation in vitro: Relative mean viability of three individual tissues after 15 minutes exposure and 42 hours post-incubation was > 50 % of the mean viability of the negative controls. Classification for skin irritation in accordance with Regulation (EC) No1272/2008 is therefore not required.

 

Eye damage/irritation in vitro: Classification in accordance with Regulation (EC) No1272/2008 is not required because the In Vitro Irritancy Score (IVIS) was determined to be 3.