3-pyridylmethanol

Endpoint summary

Description of key information

Based on the results of an OECD 439 study the test item is not requiring classification for skin irritation (UN GHS: No Category) (reference 7.3.1 -1).

Based on the results of an OECD 437 study, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (no Category) (reference 7.3.2 -1).

Based on the results of an OECD 492 study the test item is requiring classification for eye damaging potential (UN GHS Category 1 or Category 2) (reference 7.3.2 -2).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 September - 21 November 2016

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:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation 440/2008, ATP 640/2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: reconstituted human epidermis

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis, and has been validated by the ECVAM in 2008.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number(s): 16-RHE-109
- Date of initiation of testing: On day of receipt the pre-incubation phase of the tissues started.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with minimum 25 mL DPBS; excess DPBS removed by shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Observable damage in the tissue due to washing: no data
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: microplate reader ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin category 2 if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 µL

NEGATIVE CONTROL
- Amount applied: 16 µL

POSITIVE CONTROL
- Amount applied: 16 µL
- Concentration: 5%

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 triplicate tissues (3 aliquots per tissue)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1

Value:

76.26

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2

Value:

76.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3

Value:

82.35

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS
- Direct-MTT reduction: no
- Colour interference with MTT: no

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

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

Executive summary:

An in vitro study in accordance with the OECD Guideline OECD 439 was performed to assess the skin irritation potential of the test item.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes. 16 µL of either the negative control (DPBS), the positive control (5% SLS) or the test item were applied to the tissues.

The test item did not reduce MTT, and it did not indicate colour interference. All of the acceptability criteria were met. The absorbance values of the negative control were between 1.7 and 1.9. The positive control induced a sufficient decrease of the relative absorbance as compared to the negative control. The relative standard deviations of three replicates were below 18%.

As a result, the mean relative absorbance was determined to be 78.4% compared to the relative absorbance of the negative control after treatment with the test item. Therefore, the test item is not considered to possess an irritant potential to skin under the experimental conditions chosen (UN GHS: No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 May 2017 - 6 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

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:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: isolated bovine cornea

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Age of donor animals: 16-28 months
- Croneal diameter: 25 - 27 mm
- Storage, temperature and transport conditions of ocular tissue: cooled on ice
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL

Duration of treatment / exposure:

10 min

Number of animals or in vitro replicates:

3 corneas per group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded. Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, 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. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
After delivery all eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
At the end of the incubation period, the basal opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). The light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values). Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL, 10 min

POST-INCUBATION PERIOD: yes, 120 min

REMOVAL OF TEST SUBSTANCE:
After the incubation period the corneal surface was washed three times with wash medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The light transmission through the corneas was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany).
- Corneal permeability: The amount of fluorescein that crossed the cornea was measured spectrophotometrically with a microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS), IVIS = corrected opacity value + (15 x corrected permeability OD490 value)

DECISION CRITERIA:
IVIS ≤ 3 No Category (according to GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Serious eye damage, Category 1 (according to GHS)

Irritation parameter:

in vitro irritation score

Remarks:

Tissue 1

Run / experiment:

1

Value:

16.885

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

Tissue 2

Run / experiment:

1

Value:

29.895

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

Tissue 3

Run / experiment:

1

Value:

24.525

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: Yes
- Acceptance criteria met for positive control: Yes

Interpretation of results:

other: Not Category 1 (irreversible effects on the eye) based on GHS

Conclusions:

Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

Executive summary:

The objective of the present study was to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment.

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item. As negative control 0.9% sodium chloride solution and as positive control N,N-dimethylformamide was used.

Three corneas were used per group (negative control, positive control or test item group).

After a first opacity measurement of the untreated bovine corneas, 750 µL of the test item, positive or negative control were applied on the corneas and incubated for 10 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again.

After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically.

The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.7 (study acceptance criteria range: -1.2 - 5.3). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 103.3 (study acceptance criteria range: 75.3 - 120.2). Therefore, the study fulfilled the acceptance criteria.

The IVIS obtained after treatment with the test item was 23.8 and, thus higher than 3 and lower than 55. Therefore, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (no Category).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 July 2017 - 18 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: reconstructed human cornea-like epithelium

Details on test animals or tissues and environmental conditions:

Justification of the test method and considerations regarding applicability:
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

Characterisation of the test system:
- Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
- Lot No.: 27001
- Keratinocyte strain: 4F1188
- Supplier: MatTek In Vitro Life Science Laboratories

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

Duration of treatment / exposure:

30 ± 2 minutes at room temperature and 120 ± 15 minutes at 37 °C

Duration of post- treatment incubation (in vitro):

12 ± 2 minutes

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used:

Preparation:
On day of receipt, the tissues were equilibrated in their 24-well shipping container to room temperature for about 15 minutes. Afterwards the tissues were removed from the shipping container using sterile forceps and transferred to 6-well plates containing 1 mL pre-warmed (37°C) assay medium. Any agarose adhering to the inserts was removed by gentle blotting on gauze or paper towel. Afterwards, the tissues were incubated at 37°C and 5% CO2 overnight (16-24 hours) without medium exchange.

Pre-Treatment:
After the overnight incubation, the tissues were pre-wetted with 20 µL DPBS and incubated at 37°C and 5% CO2 for 30 minutes (± 2 minutes).

Exposure and Post-Treatment:
After the 30 minute DPBS pre-treatment, the liquid test item, the negative and the positive control were tested by applying 50 uL topically on the EpiOcular™ tissues. The tissues were placed back into the culture medium after dosing and incubated at 37°C and 5% CO2 for 30 ± 2 minutes.
At the end of the 30 ± 2 minutes treatment time, the positive control, negative control and the test item were removed by extensively rinsing the tissues with pre-warmed (room temperature) DPBS. Three clean beakers, containing a minimum of 100 mL each of DPBS were used per group. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the two tissues per group were rinsed simultaneously by holding the replicate inserts together by their collars using forceps. The test item or control articles were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed at least two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS at least three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material.
After rinsing, the tissues were immediately transferred in 5 mL of pre-warmed (room temperature) assay medium in a 12-well plate for 12 ± 2 minutes at room temperature.
After the 12 ± 2 minutes incubation, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert were blotted on absorbent material and transferred in 6-well plates filled with 1 mL of pre-warmed (37°C) assay medium for 120 ± 15 minutes at 37°C and 5% CO2.

- RhCE tissue construct used, including batch number:

Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 27001
Keratinocyte strain: 4F1188
Supplier: MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used: 50 µL

- Duration and temperature of exposure, post-exposure: exposure: 30 ± 2 min at 37 °C; post-exposure: 12 ± 2 min at room temperature + 120 ± 15 min at 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: No. The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties.

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength used for quantifying MTT formazan: 570 nm

- Description of the method used to quantify MTT formazan:

After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37°C and 5% CO2.
The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 6-well plate containing 2 mL isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically.
The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:

If the test item-treated tissue viability is >60.0% relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category). If the test item-treated tissue viability is <60.0% relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).

- Acceptance Criteria:
The results are acceptable if:
1. The negative control OD >0.8 and <2.5,
2. The mean relative viability of the positive control is:
a) 30 minute exposure: below 50% of control viability
b) 6 hour exposure: below 50% of control viability
3. Acceptable variability between tissue replicates: < 20 %

Irritation parameter:

other: Viability (%)

Remarks:

Tissue 1

Run / experiment:

1

Value:

20.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Viability (%)

Remarks:

Tissue 2

Run / experiment:

1

Value:

19.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct MTT reduction: no
- Colour interfrence with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1: Results

Group	Tissue 1		Tissue 2		Mean		SD	Difference between tissue replicates
		OD	Viability	OD	Viability	OD	Viability
Negative Control	1.614	100.1%	1.611	99.9%	1.613	100.0%	0.14	0.2%
Positive Control	0.119	7.4%	0.126	7.8%	0.123	7.6%	0.28	0.4%
Test item	0.325	20.1%	0.314	19.5%	0.320	19.8%	0.42	0.6%

Interpretation of results:

other: Category 1 or 2 based on GHS criteria

Conclusions:

Following treatment with the test item Art. P66807 (3-Pyridinemethanol), the mean tissue viability was 19.8% and, thus, lower than 60%, i.e. according to OECD 492 the test item is identified as potentially requiring classification and labelling according to UN GHS (Category 1 or Category 2).

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model.

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 30 (± 2) minutes. 50 µL of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. The post-treatment incubation was 12 (± 2) minutes and further 120 (± 15) minutes with fresh medium.

After treatment with the negative control (sterile deionized water) the mean OD was 1.613 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 7.6% (study acceptance criterion: <50%).Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 19.8% and, thus, lower than 60%, i.e. according to OECD 492 the test item is identified as potentially requiring classification and labelling according to UN GHS (Category 1 or Category 2).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

OECD 439

An in vitro study in accordance with the OECD Guideline OECD 439 was performed to assess the skin irritation potential of the test item.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes. 16 µL of either the negative control (DPBS), the positive control (5% SLS) or the test item were applied to the tissues.

The test item did not reduce MTT, and it did not indicate colour interference. All of the acceptability criteria were met. The absorbance values of the negative control were between 1.7 and 1.9. The positive control induced a sufficient decrease of the relative absorbance as compared to the negative control. The relative standard deviations of three replicates were below 18%.

As a result, the mean relative absorbance was determined to be 78.4% compared to the relative absorbance of the negative control after treatment with the test item. Therefore, the test item is not considered to possess an irritant potential to skin under the experimental conditions chosen (UN GHS: No Category).

Eye irritation

OECD 437

An in vitro study in accordance with the Guideline OECD 437 was performed to examine the potential of the test item to induce serious eye damage. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment.

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item. As negative control 0.9% sodium chloride solution and as positive control N,N-dimethylformamide was used.

Three corneas were used per group (negative control, positive control or test item group).

After a first opacity measurement of the untreated bovine corneas, 750 µL of the test item, positive or negative control were applied on the corneas and incubated for 10 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again.

After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically.

The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.7 (study acceptance criteria range: -1.2 - 5.3). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 103.3 (study acceptance criteria range: 75.3 - 120.2). Therefore, the study fulfilled the acceptance criteria.

The IVIS obtained after treatment with the test item was 23.8 and, thus higher than 3 and lower than 55. Therefore, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (no Category).

OECD 492

The objective of the study according to OECD guideline 492 was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model.

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 30 (± 2) minutes. 50 µL of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. The post-treatment incubation was 12 (± 2) minutes and further 120 (± 15) minutes with fresh medium.

After treatment with the negative control (sterile deionized water) the mean OD was 1.613 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 7.6% (study acceptance criterion: <50%).Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 19.8% and, thus, lower than 60%, i.e. according to OECD 492 the test item is identified as potentially requiring classification and labelling according to UN GHS (Category 1 or Category 2).

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 30 (± 2 minutes). 50 µLof the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 1.613 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 7.6% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 19.8% and, thus, lower than 60%, i.e.according to OECD 492 the test item is identified as potentially requiring classification and labelling according to UN GHS (Category 1 or Category 2).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on this data, the substance is not considered to be classified for skin irritation and considered to be classified for eye damaging potential under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.