[(9-oxo-9H-thioxanthen-2-yl)oxy]acetic acid

Endpoint summary

Description of key information

Studies conducted to GLP under recognised testing guidelines.

OECD 439

The objective of this study was to evaluate CMTX 2-carboxymethyloxy-thioxanthone for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKINSmall model (EPISKIN-SM^TM)). The possible skin irritation potential of the test item wastested through topical application for 15 minutes.

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

The test item was a yellow powder. Skin tissue was moistened with 5 µL of Milli-Q water and at least 10 mg of the test item was applied 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 mitochondrialdehydrogenase activity measured by formazan production from MTT at the end of thetreatment.

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 the test itemcompared to the negative control tissues was 98%. Since the mean relative tissue viability forthe test item was above 50% after 15 ± 0.5 minutes treatment the test item considered to benon-irritant.

The positive control had a mean cell viability of 11% after 15 ± 0.5 minutes exposure. The absolute 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 percentageviability of three tissues treated identically was < 7%, indicating that the test systemfunctioned properly.

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone 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 Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

OECD 431

The objective of this study was to evaluate CMTX 2-carboxymethyloxy-thioxanthone for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)).  The possible corrosive potential of CMTX 2-carboxymethyloxy-thioxanthone was tested through topical application for 3 minutes and 1 hour.

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

The test item was a yellow powder.  Skin tissue was moistened with 25 µL of Milli-Q water and at least 25 mg of the test item was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 5.9% 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 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 14%, indicating that the test system functioned properly.

OECD 437

The objective of this study was to evaluate the eye hazard potential of CMTX 2-carboxymethyloxy-thioxanthone 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 the test item was tested through topical application for approximately 240 minutes.

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

The test item was a yellow powder.  Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure 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 (20% (w/v) Imidazole) was 198 and 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 induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 12 after 4 hours of treatment.

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

OECD 492

The objective of this study was to evaluate the eye hazard potential of CMTX 2- carboxymethyloxy-thioxanthone. For this purpose the test item was topically applied on the Reconstructed Human EpiOcular™ Model.

The possible eye hazard potential of the test item was tested through topical application for 6 hours.

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

The CMTX 2-carboxymethyloxy-thioxanthone was a yellow powder. The test item (55.5 to 60.3 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 41% after 6 hours ± 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 difference between the percentage of viability of two tissues treated identically was less than 17%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 15%. Since the mean relative tissue viability for the test item was below 60% after 6 hours ± 15 minutes treatment it is considered to be potentially irritant or corrosive to the eye.

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Nov 2018 to 09 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

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 (incl. QA statement)

Specific details on test material used for the study:

Appearance: Yellow powder
Purity/Composition: 98.25%
Test item storage: At room temperature protected from light desiccated

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

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.

Test for Color Interference by the Test Item:
The test item was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the color interference, at least 25 mg 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 it was checked if a blue / purple color change was observed.

Test for Reduction of MTT by the Test Item:
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, at least 25 mg 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 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.
 
Test System Set Up:
Tissues:
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.
("Figure 1 - A Diagram of the Application" attached below)

DMEM (Dulbecco’s Modified Eagle’s Medium):
Supplemented DMEM, serum-free supplied by MatTek Corporation.

MTT medium:
MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

Environmental conditions:
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 64 - 87%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.2 - 36.7°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 solid test item was applied directly on top of the skin tissue. The test item was spread to match the size of the tissue.
To protect the test item from light glassware was wrapped in tin-foil.

Application/Treatment of the Test Item:
The skin tissues were kept in the refrigerator 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). The plates were incubated for approximately 2.5 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before CMTX 2-carboxymethyloxy-thioxanthone 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 CMTX 2-carboxymethyloxy-thioxanthone and two for a 1-hour exposure.
 
The skin was moistened with 25 µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and 26.6 to 55.1 mg of the solid test item 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 the TECAN Infinite® M200 Pro Plate Reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

At least 25 mg

Duration of treatment / exposure:

3 minutes for two tissues
1 hour for two other tissues

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.

Number of replicates:

2 replicates for each exposure time

Irritation / corrosion parameter:

% tissue viability

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

10

Remarks on result:

other:

Remarks:

Time point: 3 minutes

Irritation / corrosion parameter:

% tissue viability

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

5.9

Remarks on result:

other:

Remarks:

Time point: 1 hour

Other effects / acceptance of results:

CMTX 2-carboxymethyloxy-thioxanthone was checked for color 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 nor a blue / purple precipitate was 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 the test item 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 3-minute and 1-hour treatments with the test item compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with CMTX 2-carboxymethyloxy-thioxanthone compared to the negative control tissues was 98% and 99% respectively. Because the mean relative tissue viability for CMTX 2-carboxymethyloxy-thioxanthone was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment CMTX 2-carboxymethyloxy-thioxanthone 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). The mean relative tissue viability following the 1-hour exposure to the positive control was 5.9%.

In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was  14%, indicating that the test system functioned properly (Appendix 1, Table 3).

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate CMTX 2-carboxymethyloxy-thioxanthone for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)).  The possible corrosive potential of CMTX 2-carboxymethyloxy-thioxanthone was tested through topical application for 3 minutes and 1 hour.

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

The test item was a yellow powder.  Skin tissue was moistened with 25 µL of Milli-Q water and at least 25 mg of the test item was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 5.9% 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 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 14%, indicating that the test system functioned properly.

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 98% and 99%, respectively.  Because the mean relative tissue viability for CMTX 2-carboxymethyloxy-thioxanthone 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.

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Dec 2018 to 17 Dec 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance: Yellow powder
Purity/Composition: 98.25%
Test item storage: At room temperature protected from light desiccated

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other:

Irritation / corrosion parameter:

other: Mean absorption

Run / experiment:

570 nm

Value:

1.088

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

1.115

Positive controls validity:

valid

Remarks:

0.125

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

11

Other effects / acceptance of results:

CMTX 2-carboxymethyloxy-thioxanthone 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. 20151956). Because no color 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 the test item 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 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 the test item compared to the negative control tissues was 98%. 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 11%. 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 < 7%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone 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 Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

The objective of this study was to evaluate CMTX 2-carboxymethyloxy-thioxanthone for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKINSmall model (EPISKIN-SM^TM)). The possible skin irritation potential of the test item wastested through topical application for 15 minutes.

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

The test item was a yellow powder. Skin tissue was moistened with 5 µL of Milli-Q water and at least 10 mg of the test item was applied 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 mitochondrialdehydrogenase activity measured by formazan production from MTT at the end of thetreatment.

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 the test itemcompared to the negative control tissues was 98%. Since the mean relative tissue viability forthe test item was above 50% after 15 ± 0.5 minutes treatment the test item considered to benon-irritant.

The positive control had a mean cell viability of 11% after 15 ± 0.5 minutes exposure. The absolute 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 percentageviability of three tissues treated identically was < 7%, indicating that the test systemfunctioned properly.

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone 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 Harmonized System of Classification and Labeling 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

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 Oct 2018 to 23 Oct 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted October 09, 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance: Yellow powder
Purity/Composition: 98.25%, assumed 100% for testing
Test item storage: At room temperature protected from light desiccated

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 20% (w/v) Imidazole, 750 µL, supplied by Merck KGaA, Darmstadt, Germany.
- The test article was administered without a vehicle (319.8 to 342.0 mg).

Duration of treatment / exposure:

240 ± 10 minutes at 32 ± 1°C

Duration of post- treatment incubation (in vitro):

Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C

Number of animals or in vitro replicates:

3

Details on study design:

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:
Since no workable suspension of CMTX 2-carboxymethyloxy-thioxanthone in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas. To protect the test item from light glassware was wrapped in tin-foil.

Treatment of Corneas and Opacity Measurements:
The medium from the anterior compartment was removed and 750 µl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. CMTX 2-carboxymethyloxy-thioxanthone was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (319.8 to 342.0 mg).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

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 5 mg Na-fluorescein/mL cMEM solution (Sigma-Aldrich Chemie GmbH, Germany).
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 l 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

Remarks:

Mean value

Value:

4.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean opacity = -0.4

Positive controls validity:

valid

Remarks:

Mean opacity = 172

Irritation parameter:

other: Corneal permeability

Remarks:

Mean value

Value:

0.446

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean permeability = 0.019

Positive controls validity:

valid

Remarks:

Mean permeability = 1.728

Irritation parameter:

in vitro irritation score

Remarks:

Mean

Value:

12

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean In vitro Irritation score = -0.1

Positive controls validity:

valid

Remarks:

Mean In vitro Irritation score = 198

Other effects / acceptance of results:

CMTX 2-carboxymethyloxy-thioxanthone was tested as it is.

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

The individual in vitro irritancy scores for the negative controls ranged from -0.6 to 0.6. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 195 to 202 (Appendix 2, Table 5). The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with CMTX 2-carboxymethyloxy-thioxanthone showed opacity values ranging from 2.8 to 6.5 and permeability values ranging from 0.256 to 0.795. The corneas were turbid after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 7.1 to 18 after 240 minutes of treatment with CMTX 2-carboxymethyloxy-thioxanthone.

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 (20% (w/v) Imidazole) was 198 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.

CMTX 2-carboxymethyloxy-thioxanthone induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 12 after 240 minutes of treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since CMTX 2-carboxymethyloxy-thioxanthone 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 CMTX 2-carboxymethyloxy-thioxanthone 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 the test item was tested through topical application for approximately 240 minutes.

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

The test item was a yellow powder.  Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure 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 (20% (w/v) Imidazole) was 198 and 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 induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 12 after 4 hours of treatment.

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

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 Nov 2018 to 30 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted June 25, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance: Yellow powder
Purity/Composition: 98.25% (assumed 100% for testing)
Test item storage: At room temperature protected from light desiccated

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

- Test item, 50 mg.
- Negative control, 50 µL sterile Milli-Q water.
- Positive control, 50 µL Methyl Acetate.

Duration of treatment / exposure:

18 hours ± 15 minutes at 37°C

Duration of post- treatment incubation (in vitro):

180 ± 10 minutes at 37°C

Number of animals or in vitro replicates:

2

Details on study design:

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.

Test for Color Interference by the Test Item:
CMTX 2-carboxymethyloxy-thioxanthone was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the tissues during the exposure. To assess the color interference, approximately 50 mg of CMTX 2-carboxymethyloxy-thioxanthone 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. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g. Furthermore, or approximately 50 mg of CMTX 2-carboxymethyloxy-thioxanthone 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. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g.

At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® 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.

Test for Reduction of MTT by the Test Item:
CMTX 2-carboxymethyloxy-thioxanthone was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, approximately 50 mg of the 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 color 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.

Test System Set Up:
On the day of receipt the 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 Assay Medium. Assay Medium was supplied by MatTek Corporation, Ashland, USA.

(Figure 1 A Diagram of the Application - attached below)

DMEM (Dulbecco’s Modified Eagle’s Medium):
Supplemented DMEM medium, serum-free supplied by MatTek Corporation.

MTT medium:
MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent.

Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 54 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 33.7 - 36.8°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 solid test item (55.5 to 60.3 mg) was applied directly on top of the skin tissue. To protect the test item from light, tubes wrapped in tin-foil were used for test item preparations. Any residual volumes were discarded.

Application/Treatment of the Test Item:
The test was performed on a total of 2 tissues per test item together with a negative control and positive control.

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. Two tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues with 50 µL Methyl Acetate (positive control) respectively.

At least 50 mg solid was added into the 6-well plates on top of the tissues. After the exposure period with CMTX 2-carboxymethyloxy-thioxanthone (6 hours ± 15 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-labeled 12-well plate for a 25 ± 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 18 hours ± 15 minutes at 37°C.

Cell Viability Measurement:
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C.

After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

Irritation parameter:

other: Mean absorption

Run / experiment:

OD570

Value:

0.248

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean absorption = 1.682

Positive controls validity:

valid

Remarks:

Mean absorption = 0.684

Irritation parameter:

other: Mean tissue viability

Remarks:

Percentage of control

Value:

15

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean tissue viability (percentage of control) = 100

Positive controls validity:

valid

Remarks:

Mean tissue viability (percentage of control) = 41

Interference of the Test Item with the MTT Endpoint

CMTX 2-carboxymethyloxy-thioxanthone was checked for possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that the test item did not interact with the MTT endpoint.

CMTX 2-carboxymethyloxy-thioxanthone was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.0188 and 0.0401, respectively. Therefore it was concluded that the test item did not induce color interference.

Main Assay

The mean absorption at 570 nm measured after treatment with the test item 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 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues. Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 15%. Since the mean relative tissue viability for the test item was below 60% it is considered to be potentially irritant or corrosive to the eye.

The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 41%. The absolute mean OD₅₇₀ of the negative control tissues was within the laboratory historical control data range (See Appendix 3). The difference between the percentage of viability of two tissues treated identically was less than 17%, indicating that the test system functioned properly.

Interpretation of results:

other: Potentially requires classification

Conclusions:

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Executive summary:

The objective of this study was to evaluate the eye hazard potential of CMTX 2- carboxymethyloxy-thioxanthone. For this purpose the test item was topically applied on the Reconstructed Human EpiOcular™ Model.

The possible eye hazard potential of the test item was tested through topical application for 6 hours.

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

The CMTX 2-carboxymethyloxy-thioxanthone was a yellow powder. The test item (55.5 to 60.3 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 41% after 6 hours ± 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 difference between the percentage of viability of two tissues treated identically was less than 17%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 15%. Since the mean relative tissue viability for the test item was below 60% after 6 hours ± 15 minutes treatment it is considered to be potentially irritant or corrosive to the eye.

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available