Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 April 2012 - 11 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

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:

Amines,N-(C16-18 and C18 unsaturated alkyl) trimethylenedi-,diacetates.

EC Number:

800-153-0

Cas Number:

1313206-64-2

Molecular formula:

R-NH2+ -(CH2)3-NH3+, (CH3 COO-)2 where R = C16-18 and C18-unsat alkyl

IUPAC Name:

Amines,N-(C16-18 and C18 unsaturated alkyl) trimethylenedi-,diacetates.

Test material form:

liquid

Remarks:

yellow liquid

Details on test material:

Chemical registery number : 1313206-64-2
Chemical name : Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agreed by each registrant.

Test animals / tissue source

Species:

cattle

Strain:

not specified

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 CiToxLAB France: the eyes were transported to CiToxLAB France 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.

Preparation of the corneas: the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.
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.
Storage of the corneas: if the corneas were used immediately, after washing they were stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.

The prepared corneas were stored and used within 24 hours.
(Pre)Incubation T°C: 32°C
Date of experimental phase: 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 solution.

POSITIVE CONTROL:
The positive control was dependent on the physical nature of the test item:
- for non-surfactant liquids and for surfactants (10-minute treatments): 10% sodium hydroxide solution (10% NaOH),
- for non-surfactant solids (4-hour treatments): 20% imidazole solution in 0.9% NaCl (w/v).

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)

METHOD
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 and 5 minutes ± 5 minutes 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 concentration of 10% (w/v) in the vehicle (0.9% NaCl), 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. Thecorneas 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.

Interpretation: see below

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

31.6

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

No notable opaque spots or irregularities were observed on vehicle 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: 31.6.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

An IVIS of 31.6 was obtained. The test item is considered to be non damaging to the eye, but the study alone does not allow to conclude on the classification of the substance.

Executive summary:

The objective of this study was to evaluate the potential corrosive or severe irritant properties of the test item on bovine eyes.

The study was performed according to the guideline OECD 437( in vitro eye irritation) 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 and 5 minutes ± 5 minutes 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 concentration of 10% (w/v) in the vehicle (0.9% NaCl), 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 vehicle 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: 31.6.

 

The test item is considered to be non damaging to the eye. The study cannot be used alone to conclude on the hazardous properties of the substance to the eyes.