Amides, C8-10, N-(hydroxyethyl)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-04-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

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:

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

Version / remarks:

December 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted September 07, 2009

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-hydroxyethyl)decanamide; N-(2-hydroxyethyl)octanamide

EC Number:

938-458-7

Cas Number:

1443751-77-6

Molecular formula:

UVCB substance: Amides, C8-10 (even-numbered), N-hydroxyethyl

IUPAC Name:

N-(2-hydroxyethyl)decanamide; N-(2-hydroxyethyl)octanamide

Test material form:

solid: flakes

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with Lglutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneae used in the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and checked finally with a view box for defects listed above. Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring)
of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).

Test system

Vehicle:

physiological saline

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL of 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water)

Duration of treatment / exposure:

240±10 minutes

Number of animals or in vitro replicates:

triplicates

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes; at least 3 times with saline
- Time after start of exposure: 240±10 min

SCORING SYSTEM: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value); a test substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

TOOL USED TO ASSESS SCORE: opacitometer; permeability measurement with sodium fluorescein

Negative control: physiological saline
Positive control: 10% (w/v) Benzalkonium chloride in saline

Results and discussion

In vivo

Irritant / corrosive response data:

The individual in vitro irritancy scores for the negative controls ranged from 2.62 to 3.1 for the corneas. The individual positive control in vitro irritancy scores ranged from 178.35 to 264.03. The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity effects on the corneae (mean in vitro irritation score 217.33) corresponding to a classification as corrosive /severe irritant to the eye (CLP/EPA/GHS (Cat 1)).
The corneas treated with the test substance showed opacity values ranging from 8.33 to 16.33 and permeability values ranging from 0.902 to 1.957. The in vitro irritancy scores ranged from 21.86 to 45.69 after 240 minutes of treatment with the test substance.

Any other information on results incl. tables

Treatment	Mean Opacity	Mean Permeability	MeanIn vitro Irritation Score
Negative control	1.67	0.078	2.84
Positive control	217.33	0	217.33
Test substance	11.66	1.482	33.91

Relative to the negative control, the test item Amides, C8-10, N-(hydroxyethyl) caused an increase of the corneal opacity and strong permeability effects occurred. The calculated mean in vitro irritation score was 33.91. According to OECD 437 the test item could be classified as not

corrosive / not severe irritant to the eye. But the peeling of the upper cell layers of the corneae exposed to the test item when rinsing after the treatment period contradicts classifying the test item as non corrosive / non severe irritant.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 2.84). The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity effects on the corneae (mean in vitro irritation score 217.33) corresponding to a classification as corrosive /severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive to the eye (CLP/EPA/GHS (Cat 1)), if considering the results of the opacity and permeability measurement after exposure to the corneae, but the lost of the upper cell layers indicates a corrosive effect.

Remarks:

Criteria used for interpretation of results: other: CLP, EU GHS (Regulation (EC) No 1272/2008)

Conclusions:

Amides, C8-10, N-(hydroxyethyl) is considered to be not corrosive to the eye (CLP/EPA/GHS (Cat 1)), if considering the results of the opacity and permeability measurement after exposure to the corneae, but the lost of the upper cell layers indicates a corrosive effect.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Amides, C8-10, N-(hydroxyethyl) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 7 September 2009 and EU method B.47, December 2010.

The test substance was tested as 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water). Physiological saline was used as negative control, 10% (w/v) Benzalkonium chloride in saline as positive control.

The corneas were incubated with Amides, C8-10, N-(hydroxyethyl) solution f

or 240±10 min at 32±1°C. The test was performed in triplicates. After rinsing the corneas at least 3 times, opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

The positive and negative controls induced the appropriate responses.

The corneas treated with Amides, C8-10, N-(hydroxyethyl) showed opacity values ranging from 8.33 to 16.33 and permeability values ranging from 0.902 to 1.957. The in vitro irritancy scores ranged from 16 to 44. The mean in vitro irritancy score was 33.91 after 240 minutes of treatment with Amides, C8-10, N-(hydroxyethyl). But the peeling of the upper cell layers of the corneae exposed to the test item when rinsing after the treatment period period contradicts classifying the test item as non corrosive / non severe irritant.

In conclusion, according to the current study and under the experimental conditions reported,

the test item Amides, C8-10, N-(hydroxyethyl) is not corrosive to the eye (CLP/EPA/GHS (Cat 1)), if considering

the results of the opacity and permeability measurement after exposure to the corneae, but

the lost of the upper cell layers indicates a corrosive effect.