Oxo(propan-2-olato)aluminium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 04, 2017 to October 05, 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Most data for the substance is provided by read-across to the similar aluminium complex aluminum, benzoate C16-18-fatty acids complexes (CAS 94166-87-7), but the Target Substance contains isopropanol, which is released from the substance during hydrolysis, and classified as an eye irritant (CLP Regulation, Annex VI and REACH disseminated dossier), so we investigated eye irritancy of the Target Substance through experimental study.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

6 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant, non-guideline study, available as an unpublished report.

Justification for type of information:

The substance is the Source Substance of the read-across. However in this case a study was conducted on the Target Substance, because it rapidly hydrolyses giving propan-2-ol which has a classification for eye irritation in the CLP Regulation Annex VI and the REACH disseminated dossier.

Principles of method if other than guideline:

The rabbit enucleated eye test is used as a first stage in the assessment of ocular irritancy potential. The assay has undergone inter-laboratory validation and has been shown to reliably detect test items that are negligible, or moderate to severe ocular irritants.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- State of eyes: Enucleated
- Selection of eyes: Prior to enucleation, the eyes of the provisionally selected rabbits were examined for evidence of ocular irritation or defect, following application of Fluorescein Sodium drops BP (1% w/v). Corneal thickness values were recorded. Only animals whose eyes showed no evidence of ocular irritation or defect were used for testing purposes (see below).
- Enucleation of eyes: Rabbits were sacrificed and two to three drops of saline solution at approximately 32°C were immediately applied to the cornea. The eye was carefully removed, positioned in a perspex clamp and placed within the superfusion chamber, with the surface irrigated by a saline drip. The eyes were then allowed to equilibrate for approximately thirty minutes, before being re examined and the corneal thickness measured. Any eyes in which the corneal swelling was greater than 10% relative to the pre-enucleation measurement, or in which the cornea was stained with fluorescein, were rejected.
- Environmental conditions: The superfusion apparatus was set to a stable temperature of 32 ±1.5°C within the chamber and a peristaltic pump supplied saline solution at approximately 32°C at a flow rate of 0.15 to 0.4 mL/minute.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL, equivalent to approximately 71 mg of the test item, was sprinkled as evenly as possible over the surface of the cornea

Duration of treatment / exposure:

- Exposure: Ten seconds, then the test item was washed off the cornea using a minimum of 20 mL of saline solution at approximately 32°C

Observation period (in vivo):

- Assessment of corneal cloudiness: Assessment was made pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment
- Measurements for corneal thickness: Pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment
- The condition of the corneal epithelium: Approximately 60, 120, 180 and 240 minutes following treatment
- The uptake of fluorescein by the corneal epithelium: Pre-enucleation, post equilibration and approximately 240 minutes following treatment

Number of animals or in vitro replicates:

- Number of eyes: Three eyes were treated with test item, two additional eyes remained untreated for control purposes.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, the test item was washed off the cornea using a minimum of 20 mL of saline solution (approximately 32 ºC)
- Time after start of exposure: Ten seconds

SCORING SYSTEM: Assessment of corneal cloudiness was made according to the numerical evaluation adopted from Advances in Modern Toxicology: Dermatoxicology, 4th Ed, (F Marzulli and H Maibach, eds) Hemisphere Publishing Corporation, Washington DC, 1991, pp 749-815.

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein: A slit-lamp biomicroscope was used to examine the eye and the thickness of the cornea was measured using an ultrasonic pachymeter. For each enucleated eye a measurement was made at the optical centre, and at a further four locations at the apex of the cornea.
- Statistics: A mean value for corneal thickness was calculated from the values for the optical centre and the apex.

Irritation parameter:

cornea opacity score

Remarks:

The mean value is given below.

Run / experiment:

1

Value:

0

Remarks on result:

other: No corneal opacities or corneal swelling observed

Irritation parameter:

fluorescein retention score

Remarks:

Fluorescein uptake. Mean value is given below.

Run / experiment:

1

Value:

0

Remarks on result:

other: No Fluorescein uptake noted 240 minutes after treatment

Other effects / acceptance of results:

Corneal opacity: No corneal effects were noted in the test eyes or control eyes during the study period.
Corneal thickness: Corneal swelling of the test eyes during the study period was comparable to that observed in the control eyes over the same period.
Corneal condition: The condition of the corneal epithelium of the test eyes and control eyes appeared normal during the study period.
Fluorescein uptake: No fluorescein uptake was noted in the test eyes or control eyes 240 minutes after treatment.
Conclusion: Following assessment of the data for all endpoints, the test item was considered unlikely to have the potential to cause severe ocular irritancy in vivo.

Table1              Individual Scores for Corneal Opacity

 

Test Eyes

Control Eyes

Chamber Number

1

3

5

2

4

Time After Treatment (minutes)

60

120

180

240

60

120

180

240

60

120

180

240

60

120

180

240

60

120

180

240

Degree of Corneal Opacity

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Area of Corneal Opacity

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Maximum Corneal Opacity (corneal cloudiness x area)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Table2              Individual Measurements for Corneal Thickness (µm)

Test Eyes

Time After Treatment (minutes)

60

120

180

240

Corneal Position

1·

2

3

4

5

mean

1·

2

3

4

5

mean

1·

2

3

4

5

mean

1·

2

3

4

5

mean

Chamber Number

1

409

376

404

363

368

384.0

371

375

399

362

365

374.4

376

371

390

360

365

372.4

370

368

380

349

360

365.4

3

421

437

433

414

419

424.8

424

418

436

400

419

419.4

425

420

438

395

400

415.6

397

404

415

380

403

399.8

5

425

402

377

383

384

394.2

406

387

375

395

394

391.4

397

383

371

384

383

383.6

386

369

363

384

357

371.8

 

Control Eyes

Time After Treatment (minutes)

60

120

180

240

Corneal Position

1·

2

3

4

5

mean

1·

2

3

4

5

mean

1·

2

3

4

5

mean

1·

2

3

4

5

mean

Chamber Number

2

398

363

380

374

369

376.8

379

354

367

373

364

367.4

384

348

362

373

359

365.0

377

340

363

371

356

361.4

4

411

391

418

410

403

406.6

415

396

418

401

396

405.2

400

384

404

395

392

395.0

394

389

387

389

385

388.8

·=       Optical centre

Table3              Determination of Corneal Swelling (%)

Test Eyes

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

1

Post equilibration

372.4

N/A

3

Post equilibration

399.4

N/A

5

Post equilibration

368.4

N/A

60 Post treatment

384.0

3.1

60 Post treatment

424.8

6.4

60 Post treatment

394.2

7.0

120 Post treatment

374.4

0.5

120 Post treatment

419.4

5.0

120 Post treatment

391.4

6.2

180 Post treatment

372.4

0.0

180 Post treatment

415.6

4.1

180 Post treatment

383.6

4.1

240 Post treatment

365.4

0.0

240 Post treatment

399.8

0.1

240 Post treatment

371.8

0.9

 

Control Eyes

 

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

Test Eyes

Mean corneal swelling 1 hour following treatment 5.5%

Mean corneal swelling 2 hours following treatment 3.9%

Mean corneal swelling 4 hours following treatment 0.3%

2

Post equilibration

364.8

N/A

4

Post equilibration

392.0

N/A

60 Post treatment

376.8

3.3

60 Post treatment

406.6

3.7

Control Eyes

120 Post treatment

367.4

0.7

120 Post treatment

405.2

3.4

Mean corneal swelling 1 hour following treatment 3.5%

Mean corneal swelling 2 hours following treatment 2.0%

Mean corneal swelling 4 hours following treatment 0.0%

180 Post treatment

365.2

0.1

180 Post treatment

395.0

0.8

240 Post treatment

361.4

0.0

240 Post treatment

388.8

0.0

a =     % Corneal swelling = (mean corneal thickness post-treatment) – (mean corneal thickness post equilibratiom)x 100

                                               Mean corneal thickness post equilibration

N/A=  Not applicable

Table 4              Corneal Epithelium Condition

Test Eyes

Chamber Number

Time After Treatment (minutes)

60

120

180

240

1

Normal

 Normal

 Normal

  Normal

3

 Normal

 Normal

 Normal

 Normal

5

 Normal

 Normal

 Normal

 Normal

 

Control Eyes

Chamber Number

Time After Treatment (minutes)

60

120

180

240

2

 Normal

 Normal

 Normal

 Normal

4

 Normal

 Normal

 Normal

 Normal

Table 5              Individual Scores for Fluorescein Uptake (240 Minutes Post Dosing)

 

Test Eyes

Control Eyes

Chamber Number

1

3

5

2

4

Intensity of Fluorescein Uptake

0

0

0

0

0

Area of Fluorescein Uptake

0

0

0

0

0

Maximum Fluorescein Uptake

(intensity x area)

0

0

0

0

0

 

Conclusions:

No corneal effects or fluorescein uptake were noted, corneal swelling of the test eyes was comparable to the control eyes and the condition of the corneal epithelium appeared normal. Following assessment of the data for all endpoints, aluminum, benzoate C16-18-fatty acids complexes was considered unlikely to have the potential to cause severe ocular irritancy in vivo.

Executive summary:

Aluminum, benzoate C16-18-fatty acids complexes was considered unlikely to have the potential to cause severe ocular irritancy in vivo. The occular irritation potential of test item was assessed in a GLP-compliant, non-guideline study on enucleated rabbit eyes (Harlan 2013). The test item was applied to the cornea of three enucleated rabbit eyes maintained at 32°C in a superfusion chamber and maximal ocular irritation observations were recorded at 60, 120 and 240 minutes, for corneal opacity, fluorescein uptake and condition of the corneal epithelium. The study is considered reliable and relevant for use for this endpoint.

Cross-reference 2

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Principles of method if other than guideline:

Draize test.

Specific details on test material used for the study:

Test substance is a hydrolysis product of Target Substance.

Species:

rabbit

Irritation parameter:

overall irritation score

Remarks:

Study gives maximum Draize score of 37 (out of 110). Isopropanol is reported to be irritating to eyes. Harmonised classification includes Eye Irrit, Category 2.

Reversibility:

fully reversible

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

Irritating in rabbit test.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Isopropanol is reported to be irritating to eyes. Harmonised classification includes Eye Irrit, Category 2.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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:

28 July 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Oxo(propan-2-olato)aluminium

EC Number:

270-365-0

EC Name:

Oxo(propan-2-olato)aluminium

Cas Number:

68425-65-0

Molecular formula:

C3 H7 Al O2

IUPAC Name:

oxo(propan-2-olato)aluminium

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The three-dimensional RhCE tissue construct that is produced using primary human epidermal keratinocytes i.e., EpiOcular™ OCL-200. The EpiOcular™ OCL-200 RhCE tissues construct is similar to the in vivo corneal epithelium three-dimensional structure.

Test system

Amount / concentration applied:

Approximately 50 mg test item was applied (each test item treated insert) topically onto the EpiOcular™ tissues.

Duration of treatment / exposure:

The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37±1°C in an incubator with 5±1% CO2, 90±10% humidified atmosphere).

Duration of post- treatment incubation (in vitro):

After rinsing, the tissues were transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation (Post-Soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the Post-Soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37°C) Assay Medium. The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation).

Details on study design:

The irritation potential of a chemical may be predicted by measurement of its cytotoxic effect, as reflected in the MTT assay, on the EpiOcular™ (OCL-200-EIT) tissue. The EpiOcular™ can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. This test method can be used to partly replace the in vivo Rabbit eye test (OECD 405), without the need to use live animals.
The three-dimensional RhCE tissue construct that is produced using primary human epidermal keratinocytes i.e., EpiOcular™ OCL-200. The EpiOcular™ OCL-200 RhCE tissues construct is similar to the in vivo corneal epithelium three-dimensional structure. In this assay, the test item is applied to the surface of the cornea epithelial construct for a fixed period, removed, and the tissue allowed to express the resulting damage. Liquids and solids are treated with different exposure and post-exposure incubations. Two construct tissues are used for each treatment and control group. Relative tissue viability is determined against the negative control-treated constructs by the reduction of the vital dye MTT (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide). A concurrent positive control is used with each assay (Kaluzhny et al., 2011).

Results and discussion

In vitro

Results

Irritation parameter:

other: % Cell viability compared to negative control

Run / experiment:

1

Value:

18

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The test item XP 473 showed significantly reduced cell viability in comparison to the negative control (mean relative viability: 18 %). All obtained test item viability results were below 60% when compared to the viability values obtained from the negative control.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item XP 473 indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2. However, the irritancy is expected to be due to isopropanol released by hydrolysis of the test substance, classified in CLP Regulation Annex VI and the REACH disseminated dossier as Eye Irrit 2, and this is considered the maximum classification.