Aluminium triethanolate

Endpoint summary

Description of key information

Guideline in vitro skin irritation and skin corrosion assays are available on the substance as well as a guideline in vitro eye irritation assay (BCOP).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Purchased or derived from tissue obtained from accredited institutions.

Source strain:

not specified

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: Reconstructed human epidermis tissue containing normal human keratinocytes. In all cases, consent was obtained by the institutions from the donor for use of the tissues for research purposes. The cells used to create the tissues were screened for potential biological contaminants. Tissue function and barrier function tests were also conducted indicating the tissues suitability.

Justification for test system used:

Standard as per OECD guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM Tissues (0.63cm2) - strain 00267
- Tissue batch number(s): 28690
- Date of initiation of testing: 03 April 2019

Test for direct MTT reduction:
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below: 25 mg of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test item turned blue/purple relative to the control, the test item was presumed to have reduced the MTT. The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and non-viable, freeze-killed, tissues. This step was a functional check which employs freeze-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues. Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12-well plate and storing in a freezer (-14 to -30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature. In addition to the normal test procedure, the MTT reducing test item was applied to two freeze-killed tissues per exposure period. In addition, two freeze-killed tissues per exposure period remained untreated. The untreated freeze-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

Assessment of colour interference:
A test item may interfere with the MTT endpoint if it is colored or if it becomes colored when in wet or aqueous conditions. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 25 mg of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

Main test preincbation:
The assay medium was brought to room temperature before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-Minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of test item and rinsing: Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-Minute and 60-Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT-loading. Another 24-well plate was prepared for the MTT-loading. 300 µL of either pre-warmed assay medium (holding plate) or MTT medium (MTT-loading plate) was dispensed into each well. The two plates were placed into the incubator until required. After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure. For the 60-Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 25 mg of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 µL of sterile water was added for wetting of the test item to increase tissue surface contact. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period. When dosing for the 60-Minute exposure period was complete, the same procedure was repeated for the 3-Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT-loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT-loading procedure was repeated.
After the 3-Hour MTT incubation was complete, the tissue inserts were blotted and transferred to 24-well plates for formazan (reduced MTT) extraction. The formazan was extracted from the top and bottom of the tissue by completely immersing the tissue insert in 2 mL of isopropanol. The plate was covered with plate sealer, to prevent isopropanol evaporation, and stood overnight at room temperature, to allow extraction to proceed.

Absorbance/optical density measurements: After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader and LT-com analysis software.

Evaluation: The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way: Relative mean viability (%) = (mean OD570 of test item / mean OD570 of negative control) x 100

Quality criteria: Negative control: The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be = 0.8 and = 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.

Positive control: Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60-Minute positive control is < 15%.

Coefficient of variation: In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be = 30%.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of 8.0 N Potassium hydroxide

Duration of treatment / exposure:

3 minute and 60 minute exposure periods

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

Prepared in duplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

86.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

91.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Direct MTT reduction: The MTT solution containing the test item turned purple. Therefore, an assessment found the test item was able to directly reduce MTT and an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.

Assessment of colour interference: The solution containing the test item was a white color. This color was attributed to the intrinsic color of the test item itself. It was therefore unnecessary to run color correction tissues.

Quality criteria: The mean OD570 for the negative control treated tissues was 1.786 for the 3-Minute exposure period and 1.837 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 4.1% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied. In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Mean OD570 values and viabilities for the negative control item, positive control item and test item:

Tissue	Exposure period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard deviation	Coefficient of variation (%)	Relative mean viability (%)
Negative control	3 minutes	1.860	1.786	0.105	5.9	100
		1.712
	60 minutes	1.816	1.837	0.029	1.6
		1.857
Positive control	3 minutes	0.095	0.083	0.018	Na	4.6
		0.070
	60 minutes	0.084	0.075	0.013	Na	4.1
		0.066
Test item	3 minutes	1.600	1.538	0.088	5.7	86.1
		1.475
	60 minutes	1.661	1.689	0.039	2.3	91.9
		1.716

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results obtained, the test item was considered non-corrosive to skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Purchased or derived from tissue obtained from accredited institutions.

Source strain:

not specified

Details on animal used as source of test system:

SOURCE ANIMAL
- Source:
Reconstructed human epidermis tissue containing normal human keratinocytes. In all cases, consent was obtained by the institutions from the donor for use of the tissues for research purposes. The cells used to create the tissues were screened for potential biological contaminants.

The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Justification for test system used:

Standard as per OECD guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin small / human epidermis (SM/13)
- Tissue batch number(s): 20-EKIN-004
- Date of initiation of testing: 22 January 2020

Assessment of direct test item reduction of MTT:
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the following procedure: 10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

Assessment of colour interference: A test item may interfere with the MTT endpoint if it is colored or becomes colored when in contact with water. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 10 mg of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

Main test application of test item and rinsing (Day 1):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface ensuring uniform covering. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT loading/formazan extraction (Day 3): Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer (-35 to -10 ºC) for possible inflammatory mediator determination. 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 2 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/optical density measurements (Day 6): At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Data evaluation: For the test item the relative mean tissue viabilities obtained after the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way: Relative mean viability (%) = (mean OD570 of test item/mean OD570 of negative control) x 100

Acceptance criteria: The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is =40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is =18%.
Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is =0.6 and =1.5, and the SD value of the percentage viability is =18%.
Test Item: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is =18%.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 10 mg (26.3 mg/cm3)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL of SDS 5% w/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

In triplicate.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

82

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

The solution containing the test item was a white color. This color was attributed to the intrinsic color of the test item itself. It was therefore unnecessary to run color correction tissues.

The relative mean viability of the test item treated tissues was 82.0% after a 15-Minute exposure period and 42-Hour post-exposure incubation period. It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal.

Acceptance criteria: The relative mean tissue viability for the positive control treated tissues was 5.3% relative to the negative control treated tissues and the standard deviation value of the viability was 3.1%. The positive control acceptance criteria were therefore satisfied. The mean OD570 for the negative control treated tissues was 0.916 and the standard deviation value of the viability was 3.7%. The negative control acceptance criteria were therefore satisfied. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 6.6%. The test item acceptance criterion was therefore satisfied.

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item	OD570 of tissues	Mean OD570 of triplicate tissues	+/- SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	+/- SD of relative mean viability (%)
Negative control item	0.954	0.916	0.034	104.1	100	3.7
	0.906			98.9
	0.888			96.9
Positive control item	0.017	0.048	0.028	1.9	5.3	3.1
	0.056			6.1
	0.072			7.9
Test item	0.718	0.751	0.060	78.4	82	6.6
	0.715			78.1
	0.821			89.6

Interpretation of results:

GHS criteria not met

Conclusions:

In this study and under the experimental conditions reported, the test item was classified as non-irritant.

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

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)

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)

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: Abattoir local to lab
- Number of animals: Triplicate per exposure
- Characteristics of donor animals (e.g. age, sex, weight): Adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 20% w/v suspension on sodium chloride 0.9% w/v.

VEHICLE
- Concentration (if solution): The negative control, sodium chloride 0.9% w/v was used as supplied.

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

None

Number of animals or in vitro replicates:

Triplicate exposures

Details on study design:

Preparation of corneas: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of cornears andopacity reading: The medium from both chambers of each holder was replaced with fresh complete EMEM. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of corneas: The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes. At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

Application of sodium fluorescein: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability determinations: After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Opacity measurement: The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Pearmeability measurement: The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In vitro irritancy score: The following formula was used to determine the In Vitro Irritancy Score: In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value). Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

The condition of the cornea was visually assessed post treatment.

Criteria for an acceptable test:
For an acceptable test the following positive control criterion should be achieved:
20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during the previous 12 months for this testing facility.
For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values calculated from the previous 12 months data for this testing facility.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

25.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

IVIS 2.1

Positive controls validity:

valid

Remarks:

IVIS 90.2

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were cloudy post treatment. There was no evidence of tissue peeling post treatment. Residual test item was not observed after the washing phase. There was no evidence of non-uniform opacity after exposure to the test item. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity and permeability values below the established upper limits. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.

Individual and mean corneal opacity and permeability measurements:

Treatment	Cornea number	Opacity				Permeability (OD492)		IVIS
		Pre-treatment	Post-treatment	Post-treatment – pre-treatment	Corrected value	NA	Corrected value
Negative control	2	4	5	1	NA	0.001	NA	NA
	3	5	7	2	NA	0.013	NA	NA
	6	4	7	3	NA	0.002	NA	NA
	NA	NA	NA	2.0	NA	0.005	NA	2.1
Positive control	10	4	63	59	57	2.240	2.235	NA
	11	4	74	70	68	1.235	1.230	NA
	12	3	80	77	75	1.239	1.234	NA
	NA	NA	NA	NA	66.7	NA	1.566	90.2
Test item	14	3	35	32	30	0.050	0.045	NA
	15	4	34	30	28	0.014	0.009	NA
	16	4	24	20	18	0.002	0.000	NA
	NA	NA	NA	NA	25.3	NA	0.018	25.6

Interpretation of results:

study cannot be used for classification

Conclusions:

The results of the current BCOP assay are inconclusive and no prediction for eye irritation can be made.

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The values obtained in the available in vitro skin irritation studies were below the cut-offs for classification as included in the OECD test guidelines. Both studies are suitable for concluding on a lack of effects and accordingly the substance is not classified in accordance with the CLP Regulation (EC No. 1272/2008, as amended).

The available in vitro eye irritation assay (BCOP) provided an inconclusive result and accordingly cannot be used for classification purposes. As in vivo tests at REACH Annex VII are not foreseen no conclusive outcome can be made on eye irritation. However, as the skin irritation tests were conclusively negative for irritation/corrosion it is unlikely the substance would be irritant/corrosive to eyes.