3,6-bis(diethylamino)-9-[2-(methoxycarbonyl)phenyl]xanthylium tetrachlorozincate

Endpoint summary

Description of key information

Skin Corrosion: Under the conditions of this study, the non-specific colour was above acceptance criteria therefore the test material is not compatible with the test system and no conclusion can be made on the corrosive potential of the test material.

Eye Irritation: Under the conditions of this study, as the test material induced an IVIS >55, classification is required for serious eye damage.

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:

18 July 2016 to 22 July 2016

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:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method 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, kit J)
- Tissue batch number(s): Lot no.: 24306
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- Source: MatTek Corporation, Ashland MA, USA

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

CELL CULTURE
- 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 supplemented DMEM medium (Dulbecco’s Modified Eagle’s Medium, serum-free).
- MTT medium: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Environmental conditions: All incubations, with the exception of the test material incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 to 100 %, containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C.

NUMBER OF REPLICATE TISSUES: 2 per exposure time

TEST FOR THE INTERFERENCE OF THE TEST MATERIAL WITH THE MTT ENDPOINT
The test material was checked for possible colour interference before the study was started. To assess the colour interference, at least 25 mg 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.
Since the test material induced colour interference in aqueous conditions, in addition to the normal procedure, two tissues must be treated with test material for 3 minutes and two tissues for 1-hour. Instead of MTT solution these tissues will be incubated with DMEM medium.

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 was 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. A negative control, sterile Milli-Q water was tested concurrently.

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 medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM medium 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 25.2 to 28.5 mg of the solid test material was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μL Milli-Q water (negative control) and with 50 μL 8 N KOH (positive control), respectively.
In addition to the normal procedure, two tissues were treated with test material for 3 minutes and two tissues for 1-hour. Instead of MTT solution these tissues were incubated with medium. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test material, however the purple test material was incorporated into the tissue. Rinsed tissues were kept in 24 well plates on 300 μL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

CELL VIABILITY MEASUREMENT
The DMEM medium 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 overnight at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test material was classified according to remaining cell viability following exposure of the test material with either of the two exposure times.

INTERPRETATION
- Acceptability of the assay
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 to 100 % viability, the Coefficient of Variation (CV) between tissue replicates should be ≤30 %.
d) The non-specific colour should be ≤30 % relative to the negative control OD.

- Data evaluation and statistical procedures
A test material is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50 %.
b) In addition, a test 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:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50 %.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25.2 to 28.5 mg of the solid test material was added into the 6-well plates on top of the skin tissues.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL Milli-Q water

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

Duration of treatment / exposure:

3 minutes of exposure and 1 hour of exposure

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

Remarks on result:

not determinable because of methodological limitations

Other effects / acceptance of results:

The test material was checked for colour interference in aqueous conditions and for possible direct MTT reduction by adding the test material to MTT medium. As a purple colour change was observed in aqueous conditions it was concluded that the test material showed colour interference. In addition to the normal procedure, two tissues were treated with test material for 3 minutes and two tissues for 1-hour but instead of MTT solution these tissues were incubated with medium. The non-specific colour by the test material was 49 and 132 % of the negative control tissues after 3-minutes and 1 hour, respectively.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The mean tissue viability of the test material could not be calculated due to the too high non-specific colour.

ACCEPTANCE OF RESULTS
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following the 1 hour exposure to the positive control was 8 %. In the range of 20 - 100 % viability the Coefficient of Variation between tissue replicates was <14 %, indicating that the test system functioned properly.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of this study, the non-specific colour was above acceptance criteria therefore the test material is not compatible with the test system and no conclusion can be made on the corrosive potential of the test material.

Executive summary:

The potential of the test material to cause skin corrosion was assessed in an in vitro skin corrosion test using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 BIS under GLP conditions.

Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of test material was applied directly on top of the skin tissue. The test consists of topical application on the skin tissue for 3-minute and 1-hour exposure periods. After exposure the skin tissue is thoroughly rinsed to remove the test material, followed by immediate determination of the cytotoxic (corrosive) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The test material showed colour interference. In addition to the normal procedure, two tissues were treated with test material for 3 minutes and two tissues for 1-hour, however instead of MTT solution these tissues were incubated with medium. The non-specific colour by the test material was 49 and 132 % of the negative control tissues after 3-minutes and 1 hour, respectively.

The positive control had a mean relative tissue viability of 8 % after the 1 hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within 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.

Under the conditions of this study, the non-specific colour was above acceptance criteria therefore the test material is not compatible with the test system and no conclusion can be made on the corrosive potential of the test material.

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:

11 July 2016 to 12 July 2016

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:

2013

Deviations:

no

GLP compliance:

yes

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 (e.g. transport time, transport media and temperature, and other conditions): 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:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 323 to 357 mg

Duration of treatment / exposure:

240 ± 10 minutes

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects; 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 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.

NEGATIVE CONTROL USED
Yes, physiological saline

POSITIVE CONTROL USED
Yes, 20 % w/v imidazole solution prepared in physiological saline

APPLICATION DOSE AND EXPOSURE TIME
323 to 357 mg for 240 ± 10 minutes

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 µL each of the negative and positive controls were introduced onto the epithelium of the cornea. The test material was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered. 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
- Number of washing steps after exposure period: 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 (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 material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.
- Others: 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:
a) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
b) The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

478.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

445.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: permeability

Run / experiment:

mean

Value:

2.194

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The corneas treated with the test material showed opacity values ranging from 162 to 615 and permeability values ranging from 0.411 to 4.721. The corneas were turbid and purple coloured (one with a loose membrane) after the 240 minutes of treatment with the test material since it was absorbed by the cornea. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 233 to 565 after 240 minutes of treatment with the test material.
The individual in vitro irritancy scores for the negative controls ranged from 1.3 to 2.4. The individual positive control in vitro irritancy scores ranged from 139 to 200. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
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 was 159 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 both endpoints, resulting in a mean in vitro irritancy score of 478 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	1.4	0.023	1.8
Positive control	134.2	1.676	159.4
Test material	445.3	2.194	478.2

*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: Classified as Category 1 in accordance with EU criteria

Conclusions:

Under the conditions of this study, as the test material induced an IVIS >55, classification is required for serious eye damage.

Executive summary:

The hazard potential of the test material to the eye was evaluated in vitro in accordance with the standardised guideline OECD 437 under GLP conditions using the Bovine Corneal Opacity and Permeability test (BCOP test).

The eye damage of the test material was tested in isolated bovine corneas through topical application for approximately 240 minutes. The test material was added neat on top of the corneas. Concurrent negative and positive controls were run using physiological saline and 20 % imidazole solution in physiological saline, respectively.

The corneas treated with the test material showed opacity values ranging from 162 to 615 and permeability values ranging from 0.411 to 4.721. The corneas were turbid and purple coloured after the 240 minutes of treatment since the cornea absorbed the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 233 to 565 after 240 minutes of treatment with the test material.

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 159 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 both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score of 478 after 240 minutes of treatment.

Under the conditions of this study, as the test material induced an IVIS >55, classification is required for serious eye damage.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The studies below were assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Skin Corrosion

The potential of the test material to cause skin corrosion was assessed in an in vitro skin corrosion test using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 BIS under GLP conditions.

Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of test material was applied directly on top of the skin tissue. The test consists of topical application on the skin tissue for 3-minute and 1-hour exposure periods. After exposure the skin tissue is thoroughly rinsed to remove the test material, followed by immediate determination of the cytotoxic (corrosive) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The test material showed colour interference. In addition to the normal procedure, two tissues were treated with test material for 3 minutes and two tissues for 1-hour, however instead of MTT solution these tissues were incubated with medium. The non-specific colour by the test material was 49 and 132 % of the negative control tissues after 3-minutes and 1 hour, respectively.

The positive control had a mean relative tissue viability of 8 % after the 1 hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within 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.

Under the conditions of this study, the non-specific colour was above acceptance criteria therefore the test material is not compatible with the test system and no conclusion can be made on the corrosive potential of the test material.

It is considered that further in vitro testing would not offer any further insights into the possible behaviour of the test material; the properties of the material that prevented the in vitro corrosion test being suitable would also apply to the in vitro irritation test and it is therefore concluded that no further in vitro testing is warranted.

At this level of supply, it is reasoned that in vivo testing would not be an appropriate use of animals. The test material is very strongly coloured and causes staining of the skin that is deemed to be irreversible. It is therefore proposed to treat this as irreversible damage to the skin and to classify as Category 1 accordingly.

Eye Irritation

The hazard potential of the test material to the eye was evaluated in vitro in accordance with the standardised guideline OECD 437 under GLP conditions using the Bovine Corneal Opacity and Permeability test (BCOP test).

The eye damage of the test material was tested in isolated bovine corneas through topical application for approximately 240 minutes. The test material was added neat on top of the corneas. Concurrent negative and positive controls were run using physiological saline and 20 % imidazole solution in physiological saline, respectively.

The corneas treated with the test material showed opacity values ranging from 162 to 615 and permeability values ranging from 0.411 to 4.721. The corneas were turbid and purple coloured after the 240 minutes of treatment since the cornea absorbed the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 233 to 565 after 240 minutes of treatment with the test material.

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 159 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 both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score of 478 after 240 minutes of treatment.

Under the conditions of this study, as the test material induced an IVIS >55, classification is required for serious eye damage.

Justification for classification or non-classification

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