Orthoperiodic acid

Endpoint summary

Description of key information

Skin Corrosion, Eurlings (2018)

Under the conditions of this study the test material was not corrosive to the skin.

Eye Irritation, Eurlings (2018)

Under the conditions of this study, the test material induced an IVIS ≥ 55, it is concluded that the test material induces serious eye damage in the Bovine Corneal Opacity and Permeability test.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November 2017 to 01 December 2017

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:

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:

2008

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue lot number(s): 27631
- 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 (serum-free; MatTek Corporation).

EXPERIMENTAL DESIGN
- Test for the Interference of the Test Material with the MTT Endpoint: A test material may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test material is present on the tissues when the MTT viability test is performed.
- Test for Colour Interference by the Test Material: The test material was checked for possible colour interference before the study was started. Some non-coloured test materials may change into coloured materials in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, at least 25 mg of the test material 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 colour change was observed.
- Test for Reduction of MTT by the Test Material: The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test material to reduce MTT, at least 25 mg of the test material or 50 µL Milli-Q water as a negative control were added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C. At the end of the exposure time it was checked if a blue / purple colour change or a blue / purple precipitate was observed.
- MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). 

MAIN TEST
- Application/Treatment of the Test Material: 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. The plates were incubated for approximately 2.5 hours at 37.0 ± 1.0 ºC. The medium was replaced with fresh DMEM just before the test material was applied. The test was performed on a total of 4 tissues per test material together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test material and two for a 1-hour exposure. The skin was moistened with 25 µL Milli-Q water to ensure close contact of the test material to the tissue and 29.23 to 35.05 mg of the solid test material was 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.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test material. 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.

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS
- The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37 °C in air containing 5 % CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.0 °C
- Temperature of post-treatment incubation (if applicable): 37 °C with MTT

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 45 - 85 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.2 - 37.3 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity. 

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

ANALYSIS
- Calculation of Cell Viability:
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw):

ODc = ODraw – ODbl

The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:

%Viability = (ODc/mean ODlt_u+MTT) * 100

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 29.23 to 35.05 mg. No correction was made for the purity/composition of the test material.

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution): 8 N

Duration of treatment / exposure:

3 minutes or 60 minutes

Duration of post-treatment incubation (if applicable):

Incubated for 3 hours with MTT

Number of replicates:

2 per exposure time

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

86

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

23

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- The test material was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test material to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test material did not interfere with the MTT endpoint.
- Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 86 and 23 % respectively. Because the mean relative tissue viability for the test material was not below 50 % after 3 minutes treatment and not below 15 % after 1 hour treatment the test material is considered to be not corrosive. Since the stability of the test was only confirmed up to a temperature of 30 °C, it could be that some substance degraded at 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. The mean relative tissue viability following the 1-hour exposure to the positive control was 7.6 %.
- In the range of 20 – 100 % viability the Coefficient of Variation between tissue replicates was 28 % for the negative and positive control. The Coefficient of Variation between tissues replicates treated with the test material was 42 % after the 1-hour exposure. However, the individual tissue viabilities after the 1-hour exposure were 29 and 17%. Both viabilities were >15 % and therefore in the same category, indicating that the test system functioned properly.

Table 1: Mean OD570 Values and Viabilities for the Negative Control, Positive Control and Test Material

Tissue	Exposure Period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.885	1.851	0.049	3.6	100*
		1.816
	60 Minutes	2.092	1.908	0.260	18
		1.724
Positive Control	3 Minutes	0.422	0.363	0.083	28	20
		0.304
	60 Minutes	0.162	0.145	0.024	21	7.6
		0.128
Test Material	3 Minutes	1.559	1.585	0.037	3.2	86
		1.611
	60 Minutes	0.563	0.445	0.167	42	23
		0.327

OD: Optical density

* The mean percentage viability of the negative control tissue is set at 100 %.

Interpretation of results:

other: Not classified in accordance with EU Criteria

Conclusions:

Under the conditions of this study the test material was not corrosive to the skin.

Executive summary:

The potential of the test material to cause corrosion to skin was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40 bis, under GLP conditions.

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

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

The positive control had a mean relative tissue viability of 7.6 % 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 ≤28 % for the negative and positive control. The Coefficient of Variation between tissues replicates treated with the test item was 42 % after the 1-hour exposure. However, the individual tissue viabilities after the 1-hour exposure were 29 and 17 %. Both viabilities were >15 % and therefore in the same category, 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 material compared to the negative control tissues was 86 and 23%, respectively. Because the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment the test material is considered to be not corrosive.

Under the conditions of this study the test material was not corrosive to the skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November 2017 to 28 November 2017

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:

2017

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- No correction was made for the purity/composition of the test material.
- A solubility test in physiological saline was performed based on visual assessment.
- A 20 % (w/v) solution of the test material was prepared in physiological saline.
- Any residual volumes were discarded.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Bovine eyes were used as soon as possible after slaughter.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularisation by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: None reported

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration (if solution): 20 % (w/v)

VEHICLE
- Amount(s) applied: 750 µL

Duration of treatment / exposure:

240 ± 10 minutes

Duration of post- treatment incubation (in vitro):

None

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION 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 and 1 % (v/v) Foetal Bovine Serum. 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
- 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.

NUMBER OF REPLICATES
- Three corneas were selected at random for each treatment group.

VEHICLE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: 20% (w/v) Imidazole solution prepared in physiological saline.

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 240 ± 10 minutes at 32 ± 1 °C.

TREATMENT METHOD
- The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or 20 % (w/v) solution of the test material was introduced onto the epithelium of the cornea.
- 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.

REMOVAL OF TEST SUBSTANCE
- After the incubation the solutions and the test material were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies).
- Post-exposure incubation: Yes, with sodium fluorescein.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
- 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 = [(I0 / I) - 0.9894] / 0.0251

- With I0 being the empirically determined illuminance through a cornea holder but with windows and medium and I being 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 material 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 material 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.
- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Na-fluorescein 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. 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.
- After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube. 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. 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 material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.
- Possible pH effects of the test material on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Additionally the opacity and permeability values were evaluated independently to determine whether the test material induced irritation through only one of the two endpoints.

DECISION CRITERIA
- The IVIS cut-off values for identifying the test materials as inducing serious eye damage (UN GHS Category 1) and test materials not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are: In vitro score range: ≤ 3 = UN GHS No Category; > 3 but ≤ 55 = No prediction can be made; and >55 = UN GHS Category 1.

ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

236

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

RESULTS
- The individual in vitro irritancy scores for the negative controls ranged from 1.2 to 3.4.
- The individual positive control in vitro irritancy scores ranged from 139 to 206. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
- The corneas treated with the test material showed opacity values ranging from 226 to 248 and permeability values ranging from -0.026 to -0.020. The corneas were turbid and discoloured after the 240 minutes of treatment with the test material. A pH effect of the test material was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 226 to 247 after 240 minutes of treatment with the test material.

DISCUSSION
- The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20 % (w/v) Imidazole) was 168 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 material induced serious eye damage through one endpoint (opacity), resulting in a mean in vitro irritancy score of 236 after 240 minutes of treatment.

Table 1: Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score*†
Negative control	1.9	0.023	2.2
Positive control	142	1.719	168
Test Material	237	-0.022	236

* Calculated using the negative control mean opacity and mean permeability values for the positive control and test material.

† In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Interpretation of results:

other: EU Criteria Category 1, H318: Causes serious eye damage.

Conclusions:

Under the conditions of this study, the test material induced an IVIS ≥ 55, it is concluded that the test material induces serious eye damage in the Bovine Corneal Opacity and Permeability test

Executive summary:

The eye irritation potential of the test material was investigated in accordance with the standardised guideline OECD 437, under GLP conditions.

The objective of this study was to evaluate the eye hazard potential of the test material 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 material was tested through topical application for approximately 240 minutes. 

The test material was applied as a 20 % (w/v) solution (750 µL) directly on top of the corneas. 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20 % (w/v) Imidazole) was 168 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 material induced serious eye damage through one endpoint (opacity), resulting in a mean in vitro irritancy score of 236 after 4 hours of treatment.

Under the conditions of this study, the test material induced an IVIS ≥ 55, it is concluded that the test material induces serious eye damage in the Bovine Corneal Opacity and Permeability test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion, Eurlings (2018)

The potential of the test material to cause corrosion to skin was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40 bis, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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

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

The positive control had a mean relative tissue viability of 7.6 % 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≤ 28 % for the negative and positive control. The Coefficient of Variation between tissues replicates treated with the test item was 42 % after the 1-hour exposure. However, the individual tissue viabilities after the 1-hour exposure were 29 and 17 %. Both viabilities were >15 % and therefore in the same category, 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 material compared to the negative control tissues was 86 and 23%, respectively. Because the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment the test material is considered to be not corrosive.

Under the conditions of this study the test material was not corrosive to the skin.

Whilst the in vitro test did not classify the substance, the test material is known to have a low pH and therefore a precautionary classification for this substance has been used.

Eye Irritation, Eurlings (2018)

The eye irritation potential of the test material was investigated in accordance with the standardised guideline OECD 437, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to evaluate the eye hazard potential of the test material 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 material was tested through topical application for approximately 240 minutes. 

The test material was applied as a 20 % (w/v) solution (750 µL) directly on top of the corneas. 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20 % (w/v) Imidazole) was 168 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 material induced serious eye damage through one endpoint (opacity), resulting in a mean in vitro irritancy score of 236 after 4 hours of treatment.

Under the conditions of this study, the test material induced an IVIS ≥ 55, it is concluded that the test material induces serious eye damage in the Bovine Corneal Opacity and Permeability test.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance is classified as Category 1, H318: Causes serious eye damage. The substance is also classified as Category 1C, H314: Causes severe skin burns and eye damage.