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Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

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Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 December 2016 - 13 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Adopted July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: European Community (EC). Commission regulation (EC) 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
Test system:
human skin model
Remarks:
EpiDerm Skin Model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Remarks:
The solid test item was applied directly on top of the skin tissue. CH02906 was spread to match the size of the tissue.
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:EpiDerm Skin Model (EPI-200)
- Tissue batch number(s):Lot no.: 24940 kit J and K (first experiment) 24956 kit Q and R (second experiment)
- Production date: /
- Shipping date: 7/12/2016 (first experiment); 11/12/2017 (second experiment)
- Delivery date: /
- Date of initiation of testing: 8/12/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C (actual range 35.1 - 37.0°C)
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 35.1 - 37.0°C)was

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: /
- Observable damage in the tissue due to washing: one of the replicates of the 3-minutes treatment time
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/ml) was diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter:
- Filter bandwidth:
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD570 = 1.744 +- 0.093 (first experiment) and 1.407 +- 0.066 (second experiment), acceptance criteria: OD570= 1.0-3.0
- Barrier function: ET-50 = 5.21 hours (first experiment) and 8.09 hours (second experiment), acceptance criteria: 4.77-8.72hours
- Morphology:
- Contamination: Sterile based on long term antibiotic and antimycotic free culture
- Reproducibility:

NUMBER OF REPLICATE TISSUES: 4 (2 used after 3 minutes of exposure, 2 after 1 hour of exposure)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: not needed
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: /
Amount/concentration applied:
At least 25 mg of CH02906 was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline.
Duration of treatment / exposure:
3 minutes of exposure
1 hour of exposure
Number of replicates:
4
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
experiment 1: 3-minute exposure
Value:
ca. 72
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: results replicates different
Remarks:
one replicate was found to be corrosive (cell viability 45%), the other replicate non-corrosive (100% cell viability). The variation was most probably caused by problems during rinsing: the test item could not be fully removed from one replicate. Therefore, a second experiment was conducted.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
experiment 1: 1-hour exposure
Value:
ca. 83
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
experiment 2: 3-minutes exposure
Value:
ca. 104
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
experiment 2: 1-hour exposure
Value:
ca. 81
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: High variability
Remarks:
Although the variability between the two replicates was high (54%, cell viability of replicate 1 and 2 were 51% and 112%, respectively), both replicates were found to be non-corrosive.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system:/
- Direct-MTT reduction:no direct MTT-reduction
- Colour interference with MTT: no colour interference

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (0.8- Acceptance criteria met for positive control: yes (mean tissue viability <15% after 1 hour of exposure: 14% experiment 1, 7% experiment 2)
- Acceptance criteria met for variability between replicate measurements: yes for the 1-hour exposure in the first experiment (8.8%) and for the 3-minute exposure in the second experiment (19%)
- Range of historical values if different from the ones specified in the test guideline:
Interpretation of results:
GHS criteria not met
Conclusions:
It is concluded that this test was valid and that CH02906 was not corrosive in the in vitro skin corrosion test under the experimental conditions described.
Executive summary:

In vitroskin corrosion test with CH02906 using a human skin model.

This report describes the ability of CH02906 to induce skin corrosion on a human three- dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of CH02906 was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD431 and EC B.40 BIS guidelines.

Batch 151222 of CH02906 was a white to brownish powder with a purity of 99.9%. Skin tissue was moistened with 25 μl of Milli-Q water and at least 25 mg of CH02906 was applied directly on top of the skin tissue.

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 CH02906 compared to the negative control tissues was 72% and 83%, respectively. At the 3-minutes treatment time the Coefficient of Variation between tissue replicates was 55% and the results of the individual skin tissues were 45 and 100%. Therefore one tissue had a corrosive result and the other tissue was not corrosive. This variation between replicates is most probably caused by the rinsing of the skin tissues after exposure. At the 3-minutes treatment time the test item could not be completely removed from one of the duplicate tissues during the rinsing process. The corrosion study was therefore repeated in a second experiment.

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with CH02906 compared to the negative control tissues was 104% and 81%, respectively in the second experiment. Although at the 1-hour treatment time the Coefficient of Variation between tissue replicates was 54% in experiment 2, both individual skin tissues had a non-corrosive result of 51 and 112%. This non-corrosive result after 1-hour was also observed in the first experiment. Because the mean relative tissue viability for CH02906 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, CH02906 is considered to be not corrosive.

The positive control had a mean relative tissue viability of 14 and 7% after the 1-hour exposure in the first and second experiment, respectively. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within (1.046 - 1.658) the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit2.8) and the laboratory historical control data range in both experiments. In the range of 20 - 100% viability, the Coefficient of Variation between tissue replicates was20% for the control tissues, indicating that the test system functioned properly in both experiments.

Finally, it is concluded that this test was valid and that CH02906 was not corrosive in thein vitroskin corrosion test under the experimental conditions described in this report.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 February - 06 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
20 July 2012
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult donor
Source strain:
other: 09-KERA-009 + 11-KERA-002
Justification for test system used:
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 skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM
- Tissue batch number(s): 17-EKIN-009
- Production date:
- Shipping date: February 28, 2017
- Delivery date: February 28, 2017
- Date of initiation of testing: February 28, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C (actual range 35.9 - 37.2°C)
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 35.9 - 37.2°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: /
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: /
- Filter bandwidth: /
- Linear OD range of spectrophotometer: /
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 71 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: /
- Barrier function: /
- Morphology: /
- Contamination: /
- Reproducibility: /

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
no MTT interference cfr Epiderm test

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20.2 to 24.8 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µl PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µl 5% SDS
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
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.
Number of replicates:
3 tissues per test item toether with negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Value:
ca. 101
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 12 ± 6.5 %.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically (17%) was less than 18%

historical control data range of the controls were obtained by collecting all data over the period of November 2013 to November 2016:

 

Negative control

(absorption; OD570)

Positive control

(absorption; OD570)

Positive control

(viability; %)

Range

0.676 – 1.336

0.036 – 0.549

2.85 – 45.43

Mean

1.01

0.16

15.74

SD

0.16

0.10

9.22

n

155

154

163

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, CH02906 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations based on this test and the in vitro skin corrosion study (Test Facility Study No. 515446).
Executive summary:

The objective of this study was to evaluate CH02906 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 CH02906 was tested through topical application for 15 minutes.

The study procedures described in this report were based on the OECD 439 (2009) and EC 440/2008 (2012) guidelines.

Batch 151222 of CH02906 was awhite to brownish powder. Skintissue was moistened with 5 µl of Milli-Q water and at least 10 mg of CH02906 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. The relative mean tissue viability obtained after 15±0.5 minutes treatment with CH02906 compared to the negative control tissues was 101%. Since the mean relative tissue viability for CH02906 was above 50% after 15±0.5 minutes treatment, CH02906 is considered to be non-irritant.

The positive control had a mean cell viability of 12% after 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 standard deviation of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

In conclusion, CH02906 was found to be non-irritant 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
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14-15 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
Adopted July 26, 2013
Deviations:
no
GLP compliance:
yes
Species:
cattle
Details on test animals or tissues and environmental conditions:
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Number of animals:/
- Characteristics of donor animals (e.g. age, sex, weight):/
- 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:
- indication of any existing defects or lesions in ocular tissue samples: 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.
- Indication of any antibiotics used: /
Vehicle:
unchanged (no vehicle)
Remarks:
Since no workable suspension of CH02906 in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.
Amount / concentration applied:
CH02906 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (individual values 363.7, 324.2 and
363.7 mg).
Duration of treatment / exposure:
Corneas were incubated in a horizontal position for 240 +- 10 minutes at 32 +- 1°C.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND 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) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder, closed chamber method) 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
A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
SOLVENT CONTROL USED (if applicable)
POSITIVE CONTROL USED
20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.
APPLICATION DOSE AND EXPOSURE TIME
CH02906 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (individual values 363.7, 324.2 and
363.7 mg). Corneas were incubated in a horizontal position for 240  10 minutes at 32  1C.
TREATMENT METHOD: The isolated corneas were mounted in a corneal holder (one cornea per holder, closed chamber method) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies).
- POST-EXPOSURE INCUBATION: /

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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.
- Corneal permeability: After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 l of the medium from each sampling tube was transferred to a 96-well plate.The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader, serial number 1004004186). 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.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: in vitro score range: <= 3: no category, >3;<=55: no prediction possible, >55: category 1
Irritation parameter:
cornea opacity score
Run / experiment:
Mean of three replicates
Value:
ca. 115.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Mean permeability
Run / experiment:
Mean of three replicates
Value:
ca. 1.813
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of three replicates
Value:
ca. 142.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The corneas treated with CH02906 showed opacity values ranging from 50 to 235 and permeability values ranging from 0.873 to 3.577. The corneas were turbid with spots after the 240 minutes of treatment with CH02906. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 76 to 250 after 240 minutes of treatment with CH02906. The test item could not completely be removed from the third cornea showing an IVIS of 250. Since all three corneas had an IVIS≥ 55, this had no influence on the outcome of the test.

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 149 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.

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
CH02906 induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 143 after 240 minutes of treatment.
Since CH02906 induced an IVIS ≥ 55, it is concluded that CH02906 induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report and should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
Executive summary:

Evaluation of the eye hazard potential of CH02906 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 CH02906 was tested through topical application for approximately 240 minutes.

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

Batch 151222 of CH02906 was a white to brownish powder with a purity of 99.9%. Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The meanin vitroirritancy score of the positive control (20% (w/v) Imidazole) was 149 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.

CH02906 induced serious eye damage through both endpoints, resulting in a meanin vitroirritancy score of 143 after 240 minutes of treatment.

Since CH02906induced an IVIS ≥ 55, it is concluded thatCH02906 induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report and should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification