N-(2-hydroxyethyl)dodecanamide

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

9th October 2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

9.12.2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-hydroxyethyl)dodecanamide

EC Number:

205-560-1

EC Name:

N-(2-hydroxyethyl)dodecanamide

Cas Number:

142-78-9

Molecular formula:

C14H29NO2

IUPAC Name:

N-(2-hydroxyethyl)dodecanamide

Test material form:

solid

Details on test material:

Storage: Room temperature, in the dark

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Freshly isolated bovine cornea obtained from AB Schlachth of GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): At least 9-month-old donor cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were removed after slaughter, the isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: Same day
- indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) added to Styrofoam box during transportation.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL (as a suspension)
- Concentration (if solution): The test item was tested as a 20% suspension (w/v) in saline.

VEHICLE
- Amount(s) applied (volume or weight with unit): See above
- Concentration (if solution): 0.9% NaCl in deionised water
- Lot/batch no. (if required): Not reported.
- Purity: Not reported.

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

n/a

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

APPLICATION DOSE AND EXPOSURE TIME

0.75 mL applied to each cornea and incubated at 32 ± 1 °C in the water-bath for 240 minutes.

TREATMENT METHOD

Closed chamber

POST-INCUBATION PERIOD

Following rinsing, the corneas were incubated (horizontally) for 240 minutes after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The corneas were then washed once in media without phenol red
- POST-EXPOSURE INCUBATION: Following rinsing, the corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of spectrophotometer (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify) N/A

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As described in OECD 437.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

240 minutes

Value:

ca. 7.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not classified as serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for hazard classification cannot be made.

Executive summary:

In an OCED 437 (2018),  fresh bovine corneae were exposed to the test item, N-(2-hydroxyethyl)dodecanamide  for a duration of up 240 minutes. The damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability. 


The study was performed three times since the negative control did not meet the acceptance criteria in the first run (was above the historical established boundaries) and in the second experiment, the positive control did not meet the acceptance criteria since the IVIS was higher than two Standard Deviations of the Mean IVIS of the positive control of historical established boundaries. Both experiments were declared as invalid and will not be reported. A third experiment was performed. This report reflects the data of the third experiment.

After a first opacity measurement of the fresh bovine corneae (t₀), the 20% (w/v) suspension in saline of the test item N-(2-hydroxyethyl)dodecanamide, the positive, and the negative controls were applied to the different corneae and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneae and opacity was measured again (t₂₄₀).

After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

For the negative control (saline) an increase of neither opacity nor permeability of the corneae could be observed (mean IVIS = 1.24).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 107.58) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item N-(2-hydroxyethyl)dodecanamide caused an increase of the corneal opacity. The calculated mean IVIS was 7.70 (threshold for serious eye damage: IVIS> 55).According to OECD 437, no prediction for the damage hazard of the test item to the eye can be made.

In conclusion, according to the current study and under the experimental conditions reported, the test item is not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for hazard classification cannot be made.