N-methyldidecylamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2010-07-01 to 2010-07-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

no guideline followed

Deviations:

not applicable

Principles of method if other than guideline:

The method used is adapted from that described by Gautheron P. & al. (1992) Fundam. Appl. Toxicol. 18 442-449.
The principle of this evaluation is based on the measurement of two factors: the opacity and the permeability of the treated corneas. The changes in these parameters correspond to the damages induced to the tissues.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-methyldidecylamine

EC Number:

230-990-1

EC Name:

N-methyldidecylamine

Cas Number:

7396-58-9

Molecular formula:

C21H45N

IUPAC Name:

bis(decyl)(methyl)amine

Details on test material:

- Expiration date of the lot/batch: 11 February 2011
- Storage conditions of test material: at room temperature, protected from light and under nitrogen gas

Test animals / tissue source

Species:

other: isolated cornea

Strain:

other: bovine calf

Details on test animals or tissues and environmental conditions:

Origin: calf eyes were obtained from freshly slaughtered calves at the abattoir SOCAVIA, Cany Barville, France.

Reason for choice: calf corneas 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 antibiotics (Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin).

Preparation of the corneas: The corneas were prepared as quickly as possible after receipt. Each step was carried out avoiding to touch the corneas in order to not injure them.

Selection: upon arrival at CIT, all eyes were carefully examined macroscopically for defects (opacity, scratches, pigmentation, etc) and those
exhibiting any defect were discarded. The too large eyes were also discarded in order to avoid the formation of folds at the assembly of corneas in theholder. The examination was performed under a lamp and using HBSS in order to maintain the corneas moistened and shiny. Each cornea was
observed with attention, while making swivel the eye in order to see any less refringent areas under the light or any scratches.

Preparation of the selected corneas: the tissue surrounding the eyeball was carefully pulled away and the cornea was dissected such that
approximately 2 to 3 mm of sclera was present around the cornea. The isolated corneas were stored in HBSS until all corneas were dissected.

As the corneas were not used extemporaneously for a treatment, they were washed three times of 15 minutes each in HBSS plus
penicillin/streptomycin (100 units/100 µg/mL final) at room temperature, then stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C for 24 hours maximum before use.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable (control corneas were treated with the negative control)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): a volume of 750 μL ± 8 μL was gently applied to the cornea, as uniformly as possible.

Duration of treatment / exposure:

30 minutes and 4 hours

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Not applicable (three corneas were used for each treated series)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): as the dosage form was liquid, the anterior compartment of the holder was emptied using a metal gavage tube attached to a vacuum pump, then the compartment was filled with heated cMEM (32°C). In the second experiment, the dosage form having adhered to the walls of the
compartment was eliminated using a cotton bud. Then the compartment was filled with heated cMEM (32°C).
The rinsing was repeated four times.

TOOL USED TO ASSESS SCORE: opacitometer, fluorescein, spectrophotometer

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: in vitro score = Corrected Opacity + (15 x Corrected OD490 nm)

Basis:

mean

Time point:

other: 4 hours

Score:

1.3

Reversibility:

other: not applicable

Remarks on result:

other: Following the 30-minute treatment, the mean in vitro score was -0.3

Results 2

Irritation parameter:

other: corrected opacity

Basis:

mean

Time point:

other: 4 hours

Score:

0.7

Reversibility:

other: not applicable

Results 3

Irritation parameter:

other: corrected OD490 nm

Basis:

mean

Time point:

other: 4 hours

Score:

0.044

Reversibility:

other: not applicable

Irritant / corrosive response data:

Following the 30-minute treatment, the mean in vitro score was -0.3. Then following the 4-hour treatment, the mean in vitro score was 1.3.

Other effects:

No notable opaque spots or irregularities were observed on negative control corneas, either following the 30-minute treatment or following the 4-hour treatment.
Fluoresceine fixation was observed on test item-treated corneas, following both the 30 minute and 4-hour treatments. Residual test item was also noted on corneas following the 4-hour treatment.

Any other information on results incl. tables

Acceptance criteria:

For each experiment, the acceptance criteria were fulfilled:

. the individual corneal opacity values of negative controls were < 10 in both experiments,

. the individual OD_490nm values of negative control corneas were < 0.100 in both experiments,

. the solution of fluoresceine (at 5 mg/mL in DPBS) diluted 1:1000 in cMEM had OD_490nm values between 0.850 and 0.940 in both experiments,

. following the 30-minute treatment, the positive control mean in vitro score was 90.3, thus demonstrating the sensitivity of the test system under the experimental conditions of this study.

Applicant's summary and conclusion

Interpretation of results:

slightly irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: scoring table showed above (any other information on materials and methods)

Conclusions:

Under these experimental conditions, the test item is slightly irritant for the isolated calf cornea and does not required a classification according to according to CLP (Reg. n° 1272/2008/EC) and directive 67/548/EEC.

Executive summary:

The aim of this study was to evaluate the potential irritant properties of the test item for the isolated calf cornea. This study was conducted in compliance with CIT’s standard operating procedures and the principles of Good Laboratory Practices.

The corneas were obtained from the eyes of freshly slaughtered calves at the abattoir. They were mounted in the corneal holders with the endothelial side against the O-ring of the posterior half of the holder. Both compartments of the corneal holder were filled in excesswith Minimal Essential Medium Eagle completed with 1% fetal calf serum plus penicillin/streptomycin (cMEM), then the holders were preincubated for 1 hour at 32°C. Three corneas were used for each treated series (test item, positive control and negative control).

Before the treatment, a first opacity measurement was performed using an opacitometer (determining the light transmission through the center of each mounted cornea). For the treatment, the test item was used in its original form.

 

The test item was tested sequentially in two consecutive experiments.

As the mean in vitro score at the 30-minute treatment was ≤ 10, the second experiment was undertaken using a 4-hour treatment.

 At the completion of the treatment period, the test item was removed from the front opening of the anterior part of the holder and the epithelium was washed.

 Following the 30-minute treatment, the corneas were incubated for 2 hours at. At the completion of the 2-hour incubation period, the second opacity measurement was performed.

Following the 4-hour treatment, the second opacity measurement was performed immediately without any further incubation after the rinsing of the dosage form.

After the second opacity measurement, the medium was removed from both compartments of each holder. The posterior compartment was refilled with cMEM at, while the anterior compartment received 1 mL of a 5 mg/mL fluoresceine solution in Dulbecco's Phosphate-Buffered Saline (DPBS). Then, the holders were incubated vertically for 90 minutes at 32°C.

 At the end of the 90-minute incubation, the optical density of the solution from the posterior compartment of the holder was measured at 490 nm in order to determine the permeability of the cornea. Then the cornea was removed from the holder and observed for opaque spots and other irregularities.

For each experiment, the acceptance criteria were fulfilled and the study was therefore considered to be valid. No notable opaque spots or irregularities were observed on negative control corneas, either following the 30-minute treatment or following the 4 -hour treatment.

Fluoresceine fixation was observed on test item-treated corneas, following both the 30-minute and 4-hour treatments. Residual test item was also noted on corneas following the 4-hour treatment.

Following the 30-minute treatment, the mean in vitro score was -0.3. Then following the 4-hour treatment, the mean in vitro score was 1.3.

 

Under these experimental conditions and according to both mean in vitro scores of the 30‑minute and 4-hour treatments, the test item

is slightly irritant for the isolated calf cornea.