(1α,2α,5α)-2,2,6-trimethylbicyclo[3.1.1]heptan-2-ol

Endpoint summary

Description of key information

In an in vitro skin irritation study according to OECD guideline 439, the test item was identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1”, since the tissue viability after exposure and post-incubation was less or equal to 50%. In an in vitro skin corrosion study according to OECD guideline 431, the test item was non-corrosive to skin according to EU CLP and UN GHS.

 

In an in vitro eye irritation study according to OECD guideline 492, the test item was either irritating or corrosive to eyes. As the test method cannot resolve between skin irritation and corrosion, additional information for classification purposes was required. In an in vitro Bovine Corneal Opacity and Permeability Assay according to OECD guideline 437, a prediction for the damage hazard could not be made (GHS) for the test item under the experimental conditions reported.

 

Conclusion: The test item was irritating but not corrosive to skin as determined in two in vitro skin irritation/corrosion studies. The test item was irritating but not corrosive to the eyes as determined in two in vitro eye irritation/corrosion studies.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-02-15

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:

2021-06-14

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012-07-06

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

2020-04-12

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcularTM Eye Irritation Test (OCL-200-EIT) For the prediction of acute ocular irritation of chemicals: Identification of chemicals not requiring classification and labeling for eye irritation or serious eye damage

Version / remarks:

2021-02-02

Deviations:

no

Remarks:

The Pre-Test (Assessment of Colored or Staining Materials and Assessment of Direct Test Article Reduction by MTT) was performed according to the MatTek Protocol “EpiOcularTM Eye Irritation Test.

Principles of method if other than guideline:

The Pre-Test (Assessment of Colored or Staining Materials and Assessment of Direct Test Article Reduction by MTT) was performed according to the MatTek Protocol “EpiOcularTM Eye Irritation Test.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT Reconstructed Human EpiDerm model purchased from MatTek Corporation (82105 Bratislava, Slovakia)
- Tissue batch number: 36101
- Date of certificate of analysis: 2021-10-27
- Date of initiation of testing: 2021-10-12

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5°C
- Temperature of post-treatment incubation: 24 ± 2 hours at room temperature and further 18 ± 2 hours at 37 ± 1.5°C.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were gently rinsed with PBS several times
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours ± 5 minutes
- Spectrophotometer: Versamax® (Molecular Devices)
- Wavelength: OD 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Tissue viability was checked for the tissue batch by the supplier and was found to be within the acceptable range. Concurrent negative controls (NC) were used in each run to demonstrate that viability (with the NC) of the tissues were within a defined historical acceptance range (see attached background material).
- Barrier function: Barrier function was checked for the tissue batch by the supplier and was found to be within the acceptable range. Concurrent positive controls (PC) were used in each run to demonstrate that barrier function and resulting tissue sensitivity of the tissues were within a defined historical acceptance range (see attached background material).
- Contamination: Every tissue batch was checked for biological contaminants. No HIV-1 virus, Hepatitis B or C virus and no bacteria, yeast and other funi were detected.
- Reproducibility: All acceptance criteria were met (for the tissue batch and the tissue replicates used in the study), thus, reproducibility was proven.

NUMBER OF REPLICATE TISSUES: 3 tissue replicates per test item/positive and negative control treatment

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the OD of the test item in deionised water or isopropanol at 570 nm after blank correction was > 0.08 in the first pre-experiment, it was presumed to have the potential to stain the tissue.
- Fresh tissues were used to control for color interference
- N. of replicates: Two controls in duplicates run (2 deionised water treated tissues and 2 test item treated tissues)
- Method of calculation used: Data Correction Procedure I was performed (see "Any other information on materials and methods incl. tables")


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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating or corrosive to skin if the viability after 1 hour exposure and 42 hours post-incubation is equal or less than 50%.
- The test substance is considered to be non-irritating to skin if the viability after 1 hour exposure and 42 hours post-incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 ± 2 mg (39.7 mg/cm2 according to guideline)

Duration of treatment / exposure:

60 minutes (35 minutes at 37 ± 1.5°C and 5 ± 0.5% CO2 and 25 min at room temperature)

Duration of post-treatment incubation (if applicable):

42 ± 4 hours

Number of replicates:

The test item and the controls, respectively, were tested in triplicate tissues

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 3 tissue replicates

Run / experiment:

1

Value:

6.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: Yes

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of EpiDermTM SIT for regulatory purposes, technical proficiency was demonstrated by correctly predicting the proficiency chemicals listed in OECD TG 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values: See "Attached background material"

Interpretation of results:

study cannot be used for classification

Remarks:

Further testing is required to resolve between UN GHS Categories 1 and 2 and decide on the final classification of the test item.

Conclusions:

In an in vitro skin irritation study according to OECD guideline 439, the test item was identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1”, since the tissue viability after exposure and post-incubation was less or equal to 50%.

Executive summary:

This in vitro study according to OECD guideline 439 and GLP was performed to assess the skin irritation potential of the test item by means of the Human Skin Model Test. The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Therefore, additional tests with freeze-killed tissues did not have to be performed. The test item proved to dye water in the color interference pre-experiment since the OD of the test item in deionised water at 570 nm after blank correction was > 0.08. Therefore, additional tests with viable tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the main experiment. Three tissues each of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system. After treatment with the test item, the mean relative viability value was 6.90% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin. The test item is identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1”, since the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS Categories 1 and 2 and decide on the final classification of the test item.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-06-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcularTM Eye Irritation Test (OCL-200-EIT) For the prediction of acute ocular irritation of chemicals: Identification of chemicals not requiring classification and labeling for eye irritation or serious eye damage

Version / remarks:

2021-02-02

Deviations:

yes

Remarks:

The Pre-Test (Assessment of Colored or Staining Materials and Assessment of Direct Test Article Reduction by MTT) was performed according to the MatTek Protocol “EpiOcularTM Eye Irritation Test.

Qualifier:

according to guideline

Guideline:

other: MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”

Version / remarks:

2014-11-04

Deviations:

yes

Remarks:

The Pre-Test (Assessment of Colored or Staining Materials and Assessment of Direct Test Article Reduction by MTT) was performed according to the MatTek Protocol “EpiOcularTM Eye Irritation Test.

Qualifier:

according to guideline

Guideline:

other: EC Directive 2000/33/EC, OJ L136200, dated June 08, 2000, adopting the 27th time to technical progress the Dangerous Substances Directive 67/548/EEC, Annex V, part B40 and EC regulation No 440/2008 of 30 May 2008 laying down test methods

Deviations:

yes

Remarks:

The Pre-Test (Assessment of Colored or Staining Materials and Assessment of Direct Test Article Reduction by MTT) was performed according to the MatTek Protocol “EpiOcularTM Eye Irritation Test.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2019-06-18

Deviations:

yes

Remarks:

The Pre-Test (Assessment of Colored or Staining Materials and Assessment of Direct Test Article Reduction by MTT) was performed according to the MatTek Protocol “EpiOcularTM Eye Irritation Test.

Principles of method if other than guideline:

The Pre-Test (Assessment of Colored or Staining Materials and Assessment of Direct Test Article Reduction by MTT) was performed according to the MatTek Protocol “EpiOcularTM Eye Irritation Test.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT Reconstructed Human EpiDerm model purchased from MatTek Corporation (82105 Bratislava, Slovakia)
- Tissue batch number: 36123
- Date of certificate of analysis: 2022-02-16
- Date of initiation of testing: 2022-01-21

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (3 min treatment) and 37 ± 1.5°C (60 min treatment)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were gently rinsed with PBS several times
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours ± 5 minutes
- Spectrophotometer: Versamax® (Molecular Devices)
- Wavelength: OD 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Tissue viability was checked for the tissue batch by the supplier and was found to be within the acceptable range. Concurrent negative controls (NC) were used in each run to demonstrate that viability (with the NC) of the tissues were within a defined historical acceptance range (see attached background material).
- Barrier function: Barrier function was checked for the tissue batch by the supplier and was found to be within the acceptable range. Concurrent positive controls (PC) were used in each run to demonstrate that barrier function and resulting tissue sensitivity of the tissues were within a defined historical acceptance range (see attached background material).
- Contamination: Every tissue batch was checked for biological contaminants. No HIV-1 virus, Hepatitis B or C virus and no bacteria, yeast and other funi were detected.
- Reproducibility: All acceptance criteria were met (for the tissue batch and the tissue replicates used in the study), thus, reproducibility was proven.

NUMBER OF REPLICATE TISSUES: 2 tissue replicates per time point per test item/positive and negative control treatment

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test item interfered with MTT and reduced it to formazan by itself in the second pre-experiment, two additional controls in duplicates were run with the main experiment – the freeze-killed tissue controls (killed controls = KC):
• Deionised water treated freeze-killed tissues (NC_KC)
• Test item treated freeze-killed tissues (TI_KC)
- N. of replicates: Two controls in duplicates run (2 deionised water treated freeze-killed tissues and 2 test item treated freeze-killed tissues)
- Method of calculation used: Data Correction Procedure II was performed (see "Any other information on materials and methods incl. tables")


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

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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg (39.7 mg/cm2 according to guideline)

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8.0 N Potassium Hydroxide

Duration of treatment / exposure:

3 minutes and 60 minutes

Number of replicates:

2 tissue replicates per time point per test item/positive and negative control treatment

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 2 tissue replicates

Run / experiment:

2 (60 min exposure)

Value:

25.27

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 2 tissue replicates

Run / experiment:

1 (3 min treatment)

Value:

100.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Yes
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: According to OECD 431 the laboratory demonstrated technical proficiency by correctly classifying the twelve recommended Proficiency Substances (see OECD 431, Table 1). The Test Kit supplier sent the chemicals encoded to the laboratory and accredited the correct prediction of the corrosive potential of the proficiency data afterwards. The certificate of proficiency is annexed to the report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values: See "Attached background material"

Interpretation of results:

study cannot be used for classification

Remarks:

Further testing is required to resolve between UN GHS Categories 1 and 2 and decide on the final classification of the test item.

Conclusions:

In an in vitro skin corrosion study according to OECD guideline 431, the test item was non-corrosive to skin according to EU CLP and UN GHS.

Executive summary:

This in vitro study according to OECD guideline 431 and GLP was performed to assess the corrosive potential of the test item by means of the Human Skin Model Test with EpiDerm™ tissues models. The test item reduced MTT (pre-test for direct MTT reduction). Therefore, additional tests with freeze-killed tissues had to be performed. The test item did not change colour when mixed with deionised water and isopropanol (pre-test for colour interference). Consequently, additional tests with viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (Dulbecco's Phosphate-Buffered Saline (DPBS)) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (14.94%) and for the 1 hour exposure period (12.94%) thus confirming the validity of the test system and the specific batch of tissue models. After exposure of the tissues to the test item the relative absorbance value was 100.33% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was 25.27%. Both values did not reach the threshold for corrosivity which is defined to be < 50% after the 3 minutes exposure or ≥ 50% after 3 minutes exposure and < 15% after the 1 hour exposure. Therefore, the test item is considered to be not corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item is non-corrosive to skin according to EU CLP and UN GHS. The required acceptability criteria were met.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-01-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; Identification of chemicals not requiring classification and labeling for eye irritation or serious eye damage

Version / remarks:

2021-02-02

Deviations:

no

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:

2019-06-18

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µg

Duration of treatment / exposure:

6 hours at 37 ± 1.5°C

Duration of post- treatment incubation (in vitro):

25 min at room temperature (post-exposure immersion) + 18 hours at 37 ± 1.5°C (post-exposure incubation)

Number of animals or in vitro replicates:

2 tissue replicates per test item and control treatment

Details on study design:

- Details of the test procedure used
- Doses of test chemical and control substances used: Test and control substances were tested neat
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 6 hours at 37 ± 1.5°C (exposure), 25 min at room temperature (post-exposure immersion) + 18 hours at 37 ± 1.5°C (post-exposure incubation)
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
Colouring test chemicals: Two additional controls in duplicates run with the main experiment (additional viable tissues (colored controls = CC)) were used. Two tissues were treated with deionised water (NC_CC) and two tissues were treated with the test item (TI_CC). These four tissues were incubated in medium without MTT solution in the MTT assay. At the end, Data Correction Procedure I was performed.
- Number of tissue replicates used per test chemical and controls: 2 tissues (positive control, negative control, NSCliving)
- Wavelength used for quantifying MTT formazan: 570 nm (Versamax® Molecular Devices)
- Description of the method used to quantify MTT formazan: The optical density (OD570nm) was determined spectrophotometrically in duplicates by a microplate reader (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: See "Any other information on materials and methods incl. tables"
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: See "Attached background material"
- Complete supporting information for the specific RhCE tissue construct or hCE cells used: See "Attached background material"
- Reference to historical data of the RhCE tissue construct: A Certificate of Analysis was provided by the supplier showing that all specifications were within the acceptance criteria defined by the supplier.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The technical proficiency of the test system according to OECD Guideline 492 guideline recommended proficiency substances was demonstrated. A Certificate of Proficiency is available.
- Positive and negative control means and acceptance ranges based on historical data: Acceptance ranges were met, see "Attached background material
- Acceptable variability between tissue replicates for positive and negative controls: Acceptance ranges were met, see "Attached background material
- Acceptable variability between tissue replicates for the test chemical: Acceptance ranges were met, see "Attached background material

Irritation parameter:

percent tissue viability 

Run / experiment:

Mean of two indipendent runs

Value:

1.21

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: The technical proficiency of the test system according to OECD Guideline 492 guideline recommended proficiency substances was demonstrated. A Certificate of Proficiency is available.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values: See "Attached background material"

Interpretation of results:

study cannot be used for classification

Remarks:

Further testing is required to resolve between UN GHS Categories 1 and 2 and decide on the final classification of the test item.

Conclusions:

In an in vitro eye irritation study according to OECD guideline 492, the test item was either irritating or corrosive to eyes. As no prediction can be made for the test item from this result in isolation, additional information for classification purposes is required.

Executive summary:

This in vitro study was performed according to OECD guideline 492 and GLP to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Therefore, additional tests with freeze-killed tissues did not have to be performed. The test item proved to dye water in the color interference pre-experiment since the OD of the test item in deionised water at 570 nm after blank correction was > 0.08. Therefore, additional tests with viable tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the main experiment. Each 50 mg of the test item or 50 μL of the negative control (deionised water) and of the positive control (methyl acetate), were applied to each duplicate tissue for 6 hours. The mean OD of the tissue replicates treated with the negative control was > 0.8 and < 2.8, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system. The difference of relative viability between the two relating tissues was < 20 p.p. in the same run (for test item tissues, positive and negative control tissues). After treatment with the test item a mean relative viability value of 1.21% was measured compared to the mean value of the negative control. This value is below the threshold for irritancy of ≤ 60%. In conclusion, it can be stated that in this study and under the experimental conditions reported, no prediction can be made for the test item from this result in isolation and requires additional information for classification purposes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-03-14 to 2022-07-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details

Version / remarks:

1997-04

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

2010-12-09

Deviations:

no

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:

2020-06-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Number of animals: Not specified
- Characteristics of donor animals: 14 months old donor cattle
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were stored in HBSS (Hank’s Balanced Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughterhouse and during transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneas were isolated on the same day after delivery of the eyes.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) were added to the storage solution
- Selection and preparation of corneas: The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.
- Quality check of the isolated corneas: A basal opacity measurement (t0) was conducted. Only corneas with a value of the basal opacity < 7 were used.

Vehicle:

physiological saline

Remarks:

The test item was tested as a 20% suspension (w/v) in saline using sonication for 10 minutes.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL of a 20% suspension (w/v) in saline
- Concentration: 20% (w/v) in saline

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 corneas per test item treatment, negative and positive control

Details on study design:

NUMBER OF REPLICATES: 3 corneas per test item treatment, negative and positive control

NEGATIVE CONTROL USED: Negative Control 1: Saline (0.9% NaCl in deionised water); negative Control 2: Deionised water

POSITIVE CONTROL USED: Positive Control 1: 10% (w/v) Benzalkonium chloride (purity not indicated by the producer) in saline using sonication; positive Control 2: 20 % (w/v) Imidazole in saline

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL of a 20% test item suspension (w/v) in saline was applied for 240 minutes.

TREATMENT METHOD: Specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium.

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Since the test item proved difficult to remove by the rinsing method, the front cover of the holder was opened, and the cornea was carefully washed using a gentle stream of incubation medium.

- POST-EXPOSURE INCUBATION: Fresh cMEM was added into both compartments and opacity was measured (t240).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Using an opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) which was calibrated as described in the manual. The opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)
- Others: Optical evaluation of the test item treated corneas with the unaided eye revealed no visible damage.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG were used.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3 cornea replicates

Value:

30.74

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values: See "Attached background material"

Interpretation of results:

study cannot be used for classification

Remarks:

The test method according to the OECD Guideline 437 does not allow for the evaluation of eye irritation (UN GHS Category 2). Thus, it needs to be considered together with an eye irritation study (e.g. OECD guideline 492)

Conclusions:

In an in vitro Bovine Corneal Opacity and Permeability Assay according to OECD guideline 437, a prediction for the damage hazard cannot be made (GHS) for the test item under the experimental conditions reported.

Executive summary:

This in vitro study according to OECD guideline 437 and GLP was performed to assess the corneal irritation and damage potential of the test item by means of the BCOP assay using fresh bovine corneas. After a first opacity measurement of the fresh bovine corneas (t0), the 20% (w/v) inhomogeneous suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item as well as the positive and the negative controls were each applied to different corneas fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneas and opacity was measured again (t240).

After the opacity measurements, permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (saline and deion. water), neither an increase of opacity nor permeability of the corneas could be observed (saline mean IVIS = 1.24 and deion.water mean IVIS = 1.31). The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneas corresponding to a classification as serious eye damage (EU CLP/GHS Category 1) as well as the positive control imidazole. Relative to the negative control, the test item caused an increase of the corneal permeability. The calculated mean in vitro irritancy score was 30.45. According to OECD 437, a prediction for corneal irritation and damage potential cannot be made.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

WoE, in vitro skin irritation, RL1

This in vitro study according to OECD guideline 439 and GLP was performed to assess the skin irritation potential of the test item by means of the Human Skin Model Test. The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Therefore, additional tests with freeze-killed tissues did not have to be performed. The test item proved to dye water in the color interference pre-experiment since the OD of the test item in deionised water at 570 nm after blank correction was > 0.08. Therefore, additional tests with viable tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the main experiment. Three tissues each of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system. After treatment with the test item, the mean relative viability value was 6.90% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin. The test item is identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1”, since the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS Categories 1 and 2 and decide on the final classification of the test item.

 

WoE, in vitro skin corrosion, RL1

This in vitro study according to OECD guideline 431 and GLP was performed to assess the corrosive potential of the test item by means of the Human Skin Model Test with EpiDerm™ tissues models. The test item reduced MTT (pre-test for direct MTT reduction). Therefore, additional tests with freeze-killed tissues had to be performed. The test item did not change colour when mixed with deionised water and isopropanol (pre-test for colour interference). Consequently, additional tests with viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (Dulbecco's Phosphate-Buffered Saline (DPBS)) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (14.94%) and for the 1 hour exposure period (12.94%) thus confirming the validity of the test system and the specific batch of tissue models. After exposure of the tissues to the test item the relative absorbance value was 100.33% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was 25.27%. Both values did not reach the threshold for corrosivity which is defined to be < 50% after the 3 minutes exposure or ≥ 50% after 3 minutes exposure and < 15% after the 1 hour exposure. Therefore, the test item is considered to be not corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item is non-corrosive to skin according to EU CLP and UN GHS. The required acceptability criteria were met.

 

WoE, in vitro eye irritation, RL1

This in vitro study was performed according to OECD guideline 492 and GLP to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Therefore, additional tests with freeze-killed tissues did not have to be performed. The test item proved to dye water in the color interference pre-experiment since the OD of the test item in deionised water at 570 nm after blank correction was > 0.08. Therefore, additional tests with viable tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the main experiment. Each 50 mg of the test item or 50 μL of the negative control (deionised water) and of the positive control (methyl acetate), were applied to each duplicate tissue for 6 hours. The mean OD of the tissue replicates treated with the negative control was > 0.8 and < 2.8, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system. The difference of relative viability between the two relating tissues was < 20 p.p. in the same run (for test item tissues, positive and negative control tissues). After treatment with the test item a mean relative viability value of 1.21% was measured compared to the mean value of the negative control. This value is below the threshold for irritancy of ≤ 60%. In conclusion, it can be stated that in this study and under the experimental conditions reported, no prediction can be made for the test item from this result in isolation and requires additional information for classification purposes.

 

WoE, in vitro eye corrosion, RL1

An in vitro study according to OECD guideline 437 and GLP was performed to assess the corneal irritation and damage potential of the test item by means of the BCOP assay using fresh bovine corneas. After a first opacity measurement of the fresh bovine corneas (t0), the 20% (w/v) inhomogeneous suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item as well as the positive and the negative controls were each applied to different corneas fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneas and opacity was measured again (t240). After the opacity measurements, permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (saline and deion. water), neither an increase of opacity nor permeability of the corneas could be observed (saline mean IVIS = 1.24 and deion.water mean IVIS = 1.31). The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneas corresponding to a classification as serious eye damage (EU CLP/GHS Category 1) as well as the positive control imidazole. Relative to the negative control, the test item caused an increase of the corneal permeability. The calculated mean in vitro irritancy score was 30.45. According to OECD 437, a prediction for corneal irritation and damage potential cannot be made.

 

Overall conclusion: The test item was found to be irritating to skin and eyes.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item is classified and labelled for skin and eye irritation (category 2) according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.