Aluminium triethanolate

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium triethanolate

EC Number:

209-105-8

EC Name:

Aluminium triethanolate

Cas Number:

555-75-9

Molecular formula:

C2H6O.1/3Al

IUPAC Name:

aluminum triethanolate

Test material form:

solid: particulate/powder

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.