Ethyldiisopropylamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 March 2012 - 11 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EU method B.47 (In vitro eye irritation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD guideline 437 (In vitro eye irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyldiisopropylamine

EC Number:

230-392-0

EC Name:

Ethyldiisopropylamine

Cas Number:

7087-68-5

Molecular formula:

C8H19N

IUPAC Name:

ethylbis(propan-2-yl)amine

Test material form:

other: liquid

Details on test material:

- Name of test material: ETHYLDIISOPROPYLAMINE (EDIPA)
- Physical state: colorless to light yellow liquid
- Lot/batch No.: A20NCU010101
- Analytical purity: 99.74%
- Expiry date: April 2013
- Storage conditions: at room temperature and protected from humidity.

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir (SOCAVIA, Cany Barville - France, SOCAVIA, Beuvillers - France or EVA, Saint-Pierre-sur-Dives - France).
Age: bovine cattle were up to 12 months old.
Reason for choice: bovine corneas are recommended by regulatory authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from supplier to CIT: the eyes were transported to CIT at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.
Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir (EVA, Saint-Pierre-sur-Dives - France).
Age: bovine cattle were up to 12 months old.
Reason for choice: bovine corneas are recommended by regulatory authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from supplier to CIT: the eyes were transported to CIT at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.
Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, neovascularization, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature. The corneas were used immediately or within a maximum of 24 hours.

The prepared corneas were stored and used within 24 hours.

Dates of experimental phase: from 07 May 2012 to 11 May 2012.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: in vitro negative and positive controls

Duration of treatment / exposure:

Exposure period of 10 minutes, followed by rinsing.

Observation period (in vivo):

Opacity measurement:
- before treatment,
- after 2-hour incubation in water.

Permeability measurement:
- after 90-min incubation in water (and other procedures), following the 2nd opacity measurement

Number of animals or in vitro replicates:

Not applicable
Triplicate corneas for each timepoint and tested substance (test item, negative control, positive control)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Rinsing: the anterior part of the eye was emptied and then rinsed 3 times with cMEM.

NEGATIVE CONTROL:
0.9% NaCl.

POSITIVE CONTROL:
10% sodium hydroxide solution (10% NaOH).

SCORING SYSTEM/TOOL
- Opacity:
Using an opacitometer
The average change in opacity during exposure is determined. It is corrected by subtracting the average negative control value from values in positive control and test item.
- Permeability:
Using a spectrophotometer: optical density (OD) at 490 nm wavelength
The optical density is corrected by subtracting the average negative control value from values in positive control and test item.
- Scoring:
In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)

Interpretation: see below

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

66.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

In vitro, the test item was corrosive or severe irritant to the eye.

Executive summary:

The objective of this study was to evaluate the potential corrosive or severe irritant properties of ethyldiisopropylamine on bovine eyes. The study was performed according to the guideline OECD 437, in compliance with CIT standard operating procedures and with the principles of Good Laboratory Practices. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour at 32°C. Three corneas were used for each treated series (test item, positive control and vehicle control). Before the treatment, a first opacity measurement was performed using an opacitometer. The test item was tested at the concentrations of 100%, in a single experiment using a treatment time of 10 minutes. At the completion of the treatment period, the test item was removed from the front opening of the anterior chamber and the epithelium was rinsed. The corneas were then incubated for 2 hours at 32°C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluoresceine solution. The holders were then incubated vertically for 90 minutes at 32°C. At the end of the incubation, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Then the cornea was observed for opaque spots and other irregularities.

No notable opaque spots or irregularities were observed on negative control corneas following the treatment. Opacity and fluoresceine fixation were observed on the corneas treated with the test item following treatment. The In Vitro Irritancy Score (IVIS) was: 66.6. As the test item induced an IVIS = 55.1, it was considered as ocular corrosive/severe irritant.

Ethyldiisopropylamine is considered to be corrosive or severely irritant to the eye.