4-hydroxybenzophenone

Endpoint summary

Description of key information

Study carried out according to recognised testing guidelines under GLP certification.

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: Reconstructed Human Epidermis (RHE) Test Method)

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:

Identification: 4-Hydroxy-benzophenone
Appearance: White powder
Purity/Composition: 99.72%
Test item storage: At room temperature desiccated

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: 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 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.

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 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. 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 33.2 to 46.3 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The solid test item was applied directly (25 mg) on top of the skin tissue. The test item was spread to match the size of the tissue.

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

Run / experiment:

3-minute application viability (percentage of control)

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

10

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application viability (percentage of control)

Value:

112

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

5.9

Other effects / acceptance of results:

4-Hydroxy-benzophenone 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 4-Hydroxy-benzophenone compared to the negative control tissues was 99% and 112% respectively. Because the mean relative tissue viability for 4-Hydroxy-benzophenone was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment 4-Hydroxy-benzophenone 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, 4-Hydroxy-benzophenone 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 4-Hydroxy-benzophenone for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)).  The possible corrosive potential of 4-Hydroxy-benzophenone 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 4 -hydroxybenzophenone was a white 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 4-Hydroxy-benzophenone compared to the negative control tissues was 99% and 112%, respectively.  Because the mean relative tissue viability for 4-Hydroxy-benzophenone was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment 4-Hydroxy-benzophenone is considered to be not corrosive.

In conclusion, 4-Hydroxy-benzophenone 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 14 Jan 2019

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:

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. QA statement)

Specific details on test material used for the study:

Identification: 4-Hydroxy-benzophenone
Appearance: White powder
Purity/Composition: 99.72%, assumed 100% for testing
Test item storage: At room temperature desiccated

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.

The test item 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. 20151983). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that 4-Hydroxy-benzophenone 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 approximately 23 hours at 37°C (Figure 1) in both experiments. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

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

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 70 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.6°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. 4-Hydroxy-benzophenone was spread to match the size 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. The skin was moistened with 5 µL Milli-Q water (Millipore Corp., Bedford, Mass.,
USA) to ensure close contact of the test item to the tissue and the solid test item (18.90 to 31.59 mg) 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. Negative and positive controls were shared with parallel studies. All information pertaining to shared tissues are archived in the raw data.

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 approximately 67 and 68 hours in the first and second experiment, respectively. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The solid test item was applied directly (18.90 to 31.59 mg) on top of the skin tissue. 4-Hydroxy-benzophenone was spread to match the size of the tissue.

Duration of treatment / exposure:

15 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

Remarks:

OD570

Run / experiment:

First experiment

Value:

0.64

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

OD570 = 1.115

Positive controls validity:

valid

Remarks:

OD570 = 0.125

Irritation / corrosion parameter:

other: Mean absorption

Remarks:

OD570

Run / experiment:

Second experiment

Value:

0.694

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

OD570 = 1.043

Positive controls validity:

valid

Remarks:

OD570 = 0.089

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean tissue viability (percentage of control)

Run / experiment:

First experiment

Value:

57

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) = 11

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean tissue viability (percentage of control)

Run / experiment:

Second experiment

Value:

67

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) = 8.5

Interpretation of results:

study cannot be used for classification

Conclusions:

Since both tests provided inconclusive results, no prediction about the irritancy of 4-Hydroxy-benzophenone can be made in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate 4-Hydroxy-benzophenone 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 4-Hydroxy-benzophenone 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.

Skin tissue was moistened with 5 µL of Milli-Q water and atleast 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 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.In the first experiment, the relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 57%. Since theindividual values were both above and below 50% (33, 107 and 32% respectively), the test was inconclusive and repeated.

The positive control had a mean cell viability of 11% after 15 ± 0.5 minutes exposure. Theabsolute 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 percentageviability of three tissues treated with the negative or positive control was < 7%, indicating

that the test system functioned properly. The standard deviation value of the percentageviability of three tissues treated with the test item was 43% which is above the maximum of

18%, but a second experiment was performed since the viability of the test item treated tissues was spread over two different categories.

In the second experiment, the relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with 4-Hydroxy-benzophenone compared to the negative control tissues was 67%. Since the individual values were, like in the first experiment, spread over two categories (viabilities of 103, 43 and 53% respectively), the test was again inconclusive.

The positive control had a mean cell viability of 8.5% 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 with the negative or positive control was < 9%, indicating that the test system functioned properly. The standard deviation value of the percentage viability of three tissues treated with the test item was 32% which is above the maximum of18%, but both tests showed inconclusive results, therefore this has no added value to theresults of this study.

Finally, since both tests provided inconclusive results, no prediction about the irritancy of 4-Hydroxy-benzophenone can be made in the in vitro skin irritation test under the experimental conditions described in this report.

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:

key study

Study period:

22 Oct 2018 to 13 Nov 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:

Identification: 4-Hydroxy-benzophenone
Appearance: White powder
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature 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:

Negative control: 750 µL
Positive control: 750 µL
The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (305.2 to 362.9 mg)

Duration of treatment / exposure:

240 ± 10 minutes at 32 ± 1°C

Duration of post- treatment incubation (in vitro):

90 ± 5 minutes at 32 ± 1°C

Number of animals or in vitro replicates:

3

Details on study design:

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) Fetal 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 compound.
Since no workable suspension of 4-Hydroxy-benzophenone in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.

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. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (305.2 to 362.9 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 item 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 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:

First experiment - mean value

Value:

1.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean opacity = -0.4

Positive controls validity:

valid

Remarks:

Mean opacity = 172

Irritation parameter:

cornea opacity score

Run / experiment:

Second experiment - mean

Value:

0.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean opacity = 5.1

Positive controls validity:

valid

Remarks:

Mean opacity = 109

Irritation parameter:

other: Mean permeability

Run / experiment:

First experiment - mean

Value:

9.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean permeability = -0.1

Positive controls validity:

valid

Remarks:

Mean permeability = 198

Irritation parameter:

other: Mean permeability

Run / experiment:

Second experiment - mean

Value:

0.244

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean permeability = 0.007

Positive controls validity:

valid

Remarks:

Mean permeability = 2.121

Irritation parameter:

in vitro irritation score

Run / experiment:

First experiment - mean

Value:

9.9

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

Irritation parameter:

in vitro irritation score

Run / experiment:

Second experiment - mean

Value:

4.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean In vitro irritation score = 5.2

Positive controls validity:

valid

Remarks:

Mean In vitro irritation score = 140

Other effects / acceptance of results:

4-Hydroxy-benzophenone was tested as it is.

Table 1 of Appendix 1 summarizes the opacity, permeability and in vitro irritancy scores of 4-Hydroxy-benzophenone and the controls. The opacity, permeability and in vitro scores of the individual corneas are shown in Appendix 2, Table 2 - 5.

In the first experiment, 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 the test item showed opacity values ranging from 0.9 to 2.4 and permeability values ranging from 0.018 to 1.664. The corneas were translucent 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 1.2 to 26 after 240 minutes of treatment with 4-Hydroxy-benzophenone.

Since the results for 4-Hydroxy-benzophenone were spread over 2 categories (26, 2.7 and 1.2, respectively), the test was inconclusive and a repeat experiment was performed.

In the second experiment, the individual in vitro irritancy scores for the negative controls ranged from 4.1 to 6.3. 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 116 to 168 (Appendix 2, Table 5). The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with 4-Hydroxy-benzophenone showed opacity values ranging from -0.1 to 1.8 and permeability values ranging from 0.060 to 0.600. The corneas were clear 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 2.2 to 8.9 after 240 minutes of treatment with 4-Hydroxy-benzophenone.

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 (20% (w/v) Imidazole) was 198 and 140 in the first and second experiment, respectively 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 mean in vitro irritancy score was 9.9 and 4.6 after 240 minutes of treatment with the test item in the first and second experiment, respectively.

The results for 4-Hydroxy-benzophenone were spread over 2 categories in both experiments.

Interpretation of results:

study cannot be used for classification

Conclusions:

Since both tests provided inconclusive results, no conclusion can be drawn for the eye irritation or serious eye damage of 4-Hydroxy-benzophenone.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of 4-Hydroxy-benzophenone 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 4-Hydroxy-benzophenone 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 white 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.

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 (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 mean in vitro irritancy score was 9.9 after 10 minutes of treatment with the test item. Since the results were spread over 2 categories (26, 2.7 and 1.2, respectively), the test was inconclusive and a repeat experiment was performed.

In the second 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 (20% (w/v) Imidazole) was 140 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 4.6 after 10 minutes of treatment with the test item. Since the results were, like in the first experiment, spread over 2 categories (2.7, 8.9 and 2.2, respectively), the test was again inconclusive.

Finally, since both tests provided inconclusive results, no conclusion can be drawn for the eye irritation or serious eye damage of 4-Hydroxy-benzophenone.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Jan 2019 to 22 Feb 2019

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: White powder
Purity/Composition: 99.72%
Test item storage: At room temperature desiccated

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

6 hours ± 15 minutes

Duration of post- treatment incubation (in vitro):

18 hours ± 15

Number of animals or in vitro replicates:

2

Details on study design:

Test System:
EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27461 Kit A and 27466 Kit J, Appendix 4)

The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Rationale:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

Source:
MatTek Corporation, Ashland MA, U.S.A.

Experimental 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:
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 tissues during the exposure. To assess the color interference, approximately 50 mg of the test item or 50 µL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g. Furthermore, approximately 50 mg of the test item or 50 µL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking. 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:
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, 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.

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.

Freeze-killed tissues (Lot: 27446 Kit C and 16388, Appendix 4):
Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to a freezer (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them at room temperature. Further use of killed tissues was similar to living tissues.

Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 51 - 99%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 - 37.6°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 (51.2 to 67.8 mg) was applied directly on top of the skin tissue. the test item was spread to match the size of the tissue.
Any residual volumes were discarded.

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

In addition, since the test item induced color interference in aqueous conditions, two tissues were treated with test item. Instead of MTT solution these tissues were incubated with DMEM. In addition, since the test item reacted with the MTT medium, two freeze-killed tissues were treated with test item and two freeze-killed non treated tissues were used per exposure time for the cytotoxicity evaluation with MTT. Furthermore, since the test item was identified as MTT reducer and caused color interference, a third set of adapted controls was required. In this control, the test item was applied to two killed tissue replicates per exposure time which underwent the entire testing procedure but were incubated with assay medium instead of MTT solution during the MTT assay.

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 of the solid test item was added into the 6-well plates on top of the tissues. After the exposure period with the test item (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: OD570 optical density

Run / experiment:

First Experiment

Value:

0.875

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean Absorption: OD570 optical density = 1.405

Positive controls validity:

valid

Remarks:

Mean Absorption: OD570 optical density = 0.577

Irritation parameter:

other: Mean Absorption: OD570 optical density

Run / experiment:

Second Experiment

Value:

0.881

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean Absorption: OD570 optical density = 1.479

Positive controls validity:

valid

Remarks:

Mean Absorption: OD570 optical density = 0.485

Irritation parameter:

other: Mean tissue viability (percentage of control)

Run / experiment:

First Experiment

Value:

62

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

Remarks on result:

not determinable

Irritation parameter:

other: Mean tissue viability (percentage of control)

Run / experiment:

Experiment 2

Value:

60

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) = 33

Remarks on result:

not determinable

Interference of the Test Item with the MTT Endpoint

The test item was checked for possible direct MTT reduction by adding the test item to MTT medium.

The test item 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 0.1494 and 0.0053, respectively. Therefore it was concluded that the test item induced color interference.

In addition, because a color change was observed in the presence of MTT it was concluded that the test item interacted with the MTT endpoint.

Since the test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), In addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was -0.71% and -0.14% of the negative control tissues in the first and second experiment, respectively. Since the %NSMTT ≤ 0.0, no correction was made for interference of the test item.

Since the test item showed color interference in aqueous conditions. In addition to the normal procedure, two tissue were treated with test item. Instead of MTT solution these tissues wereincubated with assay medium. The non-specific color of the test item was 0.20% and 0.05%of the negative control tissues in the first and second experiment respectively. The OD of thetreated tissues without MTT assay were subtracted from the ODs of the test item treatedviable tissues with MTT assay.

Since the test item both interacted with MTT and showed color interference, in addition to the normal procedure, two freeze-killed tissues were treated with test item. Instead of MTTsolution these tissues were incubated with assay medium. The non-specific color in killedtissues was -0.06% of the negative control tissues in the second experiment only. This part ofthe study was not performed in the first experiment due to a technical error (see Study plandeviation). The OD of the freeze-killed treated tissues without MTT assay was not added tothe ODs of the test item treated viable tissues with MTT assay.

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 30 ± 2 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.

In the first experiment, the positive control had a mean cell viability after 30 ± 2 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.

The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test item compared to the negative control tissues was 62%. Since the individual values were both above and below 60% (58 and 67%, respectively) the test was inconclusive and a repeat experiment was performed.

In the second experiment, the positive control had a mean cell viability after 30 ± 2 minutes exposure of 33%. 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 8% for the positive control and test item treated tissues.

The difference between the percentage of viability of two tissues treated with the negative control was 33% which is above the acceptance criteria of 20%. Since all individual viabilities were >50% and the absolute mean OD₅₇₀ (optical density at 570 nm) of the two tissues of the negative control were within the laboratory historical control data range, this does not influence the outcome of the test. (See Study Plan Deviation).

The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 60% (56 and 63%, respectively). Since theindividual values were, like in the first experiment, spread over two categories, the test wasagain inconclusive.

In conclusion, since both tests provided inconclusive results, no conclusion can be drawn for the EpiOcular™ test.

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, 4-Hydroxy-benzophenone is inconclusive in the EpiOcular™ test.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of 4-Hydroxy-benzophenone. 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 test item was a white powder. The test item (51.2 to 67.8 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.

In the first experiment, 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.

The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

In addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissue were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT (%NSMTT) by the test item was -0.71 % of the negative control tissues. Since the %NSMTT ≤ 0.0, no correction was made for interference of the test item.

The test item showed color interference in aqueous conditions. In addition to the normal procedure, two tissue were treated with test item. Instead of MTT solution these tissues were incubated with assay medium. The non-specific color of the test item was 0.20% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay.

For the true tissue viability to be calculated, in addition to the normal procedure, two freeze-killed tissues treated with test item had to be measured but instead of MTT solution these tissues should have been incubated with assay medium. This part of the study was not performed (see Study plan deviation).

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 62%. Since the individual values were both above and below 60% (58 and 67%, respectively) the test was inconclusive and a repeat experiment was performed.

In the second experiment, the positive control had a mean cell viability of 33% 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 8% for the positive control and test item treated tissues.

The difference between the percentage of viability of two tissues treated with the negative control was 33% which is above the acceptance criteria of 20%. Since all individual viabilities were >50% and the absolute mean OD₅₇₀ (optical density at 570 nm) of the two tissues of the negative control were within the laboratory historical control data range, this does not influence the outcome of the test. (See Study Plan Deviation).

The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

In addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissue were used for the cytotoxicity evaluation with MTT. The non-specific reduction of %NSMTT by the test item was -0.14% of the negative control tissues. Since the %NSMTT ≤ 0.0, no correction was made for interference of the test item.

The test item showed color interference in aqueous conditions. In addition to the normal procedure, two tissue were treated with test item. Instead of MTT solution these tissues were incubated with assay medium. The non-specific color of the test item was 0.05% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay.

For the true tissue viability to be calculated, in addition to the normal procedure, two freeze-killed tissues treated with test item were measured but instead of MTT solution these tissues were incubated with assay medium. The nonspecific color in freeze-killed tissues by the test item was -0.06% of the negative control tissues and no correction was made.

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 60% (56 and 63%, respectively). Since the individual values were, like in the first experiment, spread over two categories, the test was again inconclusive.

Finally, since both tests provided inconclusive results, no conclusion can be drawn for the EpiOcular™ test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available