2-ethylhexyl nitrate

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:

other: compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl nitrate

EC Number:

248-363-6

EC Name:

2-ethylhexyl nitrate

Cas Number:

27247-96-7

Molecular formula:

C8H17NO3

IUPAC Name:

2-ethylhexyl nitrate

Test animals / tissue source

Species:

other: isolated calf 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). For the transport, a container with smooth internal surfaces was used to avoid damage to the corneas.

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 the holder. 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

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:

Three corneas were used for each treated series (test item, positive control and negative control).

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the completion of the treatment period, the test item (or positive control or negative control) was removed from the front opening of the anterior part of the holder and the epithelium was washed. 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). The rinsing was repeated three times.

TOOL USED TO ASSESS SCORE: opacitometer, fluorescein and spectrophotomter.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

In vivo

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.
No notable opaque spots or irregularities were observed on test item-treated corneas, following the 30-minute treatment while fluoresceine fixation was noted on corneas, following the 4 hour treatment.

Any other information on results incl. tables

For each experiment, the acceptancecriteria were fulfilled:

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

.           the individual OD_{490 nm}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_{490 nm} values between 0.850 and 0.940 inb oth experiments,

.           following the 30-minute treatment, the positive control mean in vitro scores was 124.1, 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 (see section "any other information on materials and methods")

Conclusions:

Under the experimental conditions of this study, according to both mean in vitro scores of the 30 minute and 4-hour treatments, the test item 2-Ethylhexyl nitrate tested in its original form is classified as slightly irritant for the isolated calf cornea.

Executive summary:

Method

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 excess with 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 undiluted.

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 32°C. 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.

Results

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. 

No notable opaque spots or irregularities were observed on test item-treated corneas, following the 30-minute treatment while fluoresceine fixation was noted on corneas, following the 4‑hour treatment.

 

Following the 30-minute treatment, the mean in vitro score was 3.0. Then following the 4‑hour treatment, the mean in vitro score was 6.2.

 

Conclusion

Under the experimental conditions of this study, according to both mean in vitro scores of the 30‑minute and 4-hour treatments, the test item 2-Ethylhexyl nitrate tested in its original form is classified as slightly irritant for the isolated calf cornea.