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Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 May 2018 to24 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
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 by EC No. 640/2012 OJ No. L193, 20 July 2012.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: Photomer 4250
Appearance: Colourless to pale yellow liquid
Purity/Composition: 100% (UVCB)
Test item storage: At room temperature protected from light
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: 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
Vehicle:
unchanged (no vehicle)
Details on test system:
Test for the Interference of the Test Item with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored 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.

Photomer 4250 was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20142197). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that Photomer 4250 did not interfere with the MTT endpoint.

Test System Set Up:
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 21 hours at 37°C (Figure 1). 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 - 100% (actual range 59 - 84%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.2°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. Twenty five µL of the undiluted test item 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 68 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Amount/concentration applied:
The liquid test item was applied undiluted (25 µL) directly on top of the tissue.
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
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.
Number of replicates:
3 replicates
Irritation / corrosion parameter:
other: Mean absorption
Value:
0.074
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Basis: other: Optical density Remarks: Negative control = 1.003; Positive control = 0.046
Irritation / corrosion parameter:
other: Mean tissue viability
Value:
7.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
Basis: other: percentage of control. Remarks: Negative control = 100%; Positive control = 4.6%. (migrated information)
Other effects / acceptance of results:
Photomer 4250 was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20142197). Because no color changes were observed it was concluded that Photomer 4250 did not interact with the MTT endpoint.

The mean absorption at 570 nm measured after treatment with Photomer 4250 and controls are presented in Appendix 1, Table 1.

The individual OD570 measurements are presented in Appendix 2.

Table 2 shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with Photomer 4250 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 Photomer 4250 compared to the negative control tissues was 7.4%. Since the mean relative tissue viability for Photomer 4250 was below 50% it is considered to be irritant.

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 4.6%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See Appendix 3). The standard deviation value of the percentage viability of three tissues treated identically was < 4%, indicating that the test system functioned properly.
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In conclusion, Photomer 4250 is irritant in the in vitro skin irritation test under the experimental conditions described in this report and should be classified category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
Executive summary:

The objective of this study was to evaluate Photomer 4250 for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)).  The possible skin irritation potential of Photomer 4250 was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Photomer 4250 was a colourless to pale yellow liquid.  Photomer 4250 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 is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

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 Photomer 4250 compared to the negative control tissues was 7.4%.  Since the mean relative tissue viability for Photomer 4250 was below 50% after 15 ± 0.5 minutes treatment it is considered to be irritant.

The positive control had a mean cell viability of 4.6% 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 < 4%, indicating that the test system functioned properly.

In conclusion, Photomer 4250 is irritant in the in vitro skin irritation test under the experimental conditions described in this report and should be classified category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
08 March 2018 to 28 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
Adopted October 09, 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Appearance: Colourless to pale yellow liquid
Purity/Composition: 100% (UVCB)
Test item storage: At room temperature

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:

Specification
Corneas from bovine eyes were obtained from a local abattoir. The eyes were removed after slaughter, completely immersed in physiological saline in a suitably sized container and transported on the same day to the testing facility.

Assessment on Arrival
On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

Excision and Preparation of Corneas
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.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The negative control substance was physiological saline, 750 µL, supplied by Eurovet Animal Health, Bladel, The Netherlands
The positive control substance was ethanol, 750 µL
The test article was administered without dilution.
Duration of treatment / exposure:
120 ± 10 minutes at 32±1°C.
Duration of post- treatment incubation (in vitro):
Corneas were incubated in a horizontal position for 10±1 minutes at 32±1°C
Number of animals or in vitro replicates:
3
Details on study design:
In total 2 experiments were performed. 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.

Test Item Preparation:
No correction was made for the purity/composition of the test item. The test item was tested neat.

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 the 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 the 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 according to:

Opacity=(I_0/I-0.9894)/0.0251

With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and 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.


Irritation parameter:
cornea opacity score
Run / experiment:
Experiment 1 Mean Value
Value:
ca. 2.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Mean Opacity 2.2
Positive controls validity:
valid
Remarks:
Mean Opacity 21
Remarks on result:
not determinable
Irritation parameter:
other: Corneal Permeability
Run / experiment:
Experiment 1, Mean Value
Value:
ca. -0.046
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Mean Permeability 0.061
Positive controls validity:
valid
Remarks:
Mean Permeability 2.903
Remarks on result:
not determinable
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment 2 Mean Value
Value:
ca. 3.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Mean opacity 2.1
Positive controls validity:
valid
Remarks:
Mean opacity 23
Remarks on result:
not determinable
Irritation parameter:
other: Corneal Permeability
Run / experiment:
Experiment 2, Mean Value
Value:
ca. 0.089
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Mean Permeability 0.010
Positive controls validity:
valid
Remarks:
Mean Permeability 2.768
Remarks on result:
not determinable
Other effects / acceptance of results:
RESULTS:
Photomer 4250 was tested neat.

Table 1 of Appendix 1 summarizes the opacity, permeability and in vitro irritancy scores of Photomer 4250 and the controls. The opacity, permeability and in vitro scores of the individual corneas are shown in Table 2 - 5.

In the first experiment, the individual in vitro irritancy scores for the negative controls ranged from 2.5 to 3.8. The individual positive control in vitro irritancy scores ranged from 60 to 69 (Appendix 2,
Table 5). The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with Photomer 4250 showed opacity values ranging from 1.0 to 4.1 and permeability values ranging from -0.059 to -0.024. The corneas were translucent after the 10 minutes of treatment with Photomer 4250. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.2 to 3.8 after 10 minutes of treatment with Photomer 4250.
Since the results were spread over 2 categories (3.8, 2.7 and 0.2), the test was inconclusive and a repeat experiment was performed.

In the second test, the individual in vitro irritancy scores for the negative controls ranged from 2.0 to 2.6. The individual positive control in vitro irritancy scores ranged from 55 to 75 (Appendix 2, Table 5). The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with Photomer 4250 showed opacity values ranging from 2.7 to 4.1 and permeability values ranging from -0.018 to 0.263. The corneas were translucent after the 10 minutes of treatment with Photomer 4250. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 3.4 to 6.7 after 10 minutes of treatment with Photomer 4250.

DISCUSSION:
The mean 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 in both experiments 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.

In the first experiment, the mean in vitro irritancy score was 2.2 after 4 hours of treatment with the test item. The results were spread over 2 categories (3.8, 2.7 and 0.2 respectively).

In the second experiment, Photomer 4250 induced ocular irritation through one endpoint, resulting in a mean in vitro irritancy score of 4.8 after 10 minutes of treatment.

Over the two assays, Photomer 4250 had a mean in vitro irritancy score of 3.5. Furthermore, Photomer 4250 induced an IVIS > 3 ≤ 55 in 4 out of 6 cornea’s, therefore no prediction on the classification can be made.
Interpretation of results:
study cannot be used for classification
Conclusions:
In conclusion, since Photomer 4250 induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of Photomer 4250 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).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas.  The eye damage of Photomer 4250 was tested through topical application for 10 minutes.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 17C13003 of Photomer 4250 was a colourless to pale yellow liquid.  The test item was applied as it is (750 µL) directly on top of the corneas.

In the first experiment, the mean 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 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 mean in vitro irritancy score was 2.2 after 4 hours of treatment with the test item.

Since the results were spread over 2 categories (3.8, 2.7 and 0.2 respectively), the test was inconclusive and a repeat experiment was performed.

In the second experiment, 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 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.  

Photomer 4250 induced ocular irritation, resulting in a mean in vitro irritancy score of 4.8 after 10 minutes of treatment.

Over the two assays, Photomer 4250 had a mean in vitro irritancy score of 3.5. Furthermore, Photomer 4250 induced an IVIS > 3 ≤ 55 in 4 out of 6 cornea’s, therefore no prediction on the classification can be made.

In conclusion, since Photomer 4250 induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Additional information

Justification for classification or non-classification

The result of the eye irritation study was inconclusive and thus cannot be used for classification purposes.