Dodecanoic acid, 3-[[2-(2-hydroxyethoxy)ethyl]imino]-2,2-dimethylpropyl ester

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-03 to 2009-06-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Environmental Protection Agency. 1996. Label Review Manual: 2nd Edition. EPA737-B-96-001. Washington, DC: U.S. Environmental Protection Agency

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: European Union. 2001. General Classification and Labelling Requirements for Dangerous Substances and Preparations. In Directive 2001/59/EC (28th adaption); Annex 6 of Council Directive 67/548/EEC on the approximation of the laws, regulations and administr

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-{[2-(2-hydroxyethoxy)ethyl]imino}-2,2-dimethylpropyl dodecanoate

EC Number:

700-067-2

Cas Number:

931419-77-1

Molecular formula:

C21H41NO4

IUPAC Name:

3-{[2-(2-hydroxyethoxy)ethyl]imino}-2,2-dimethylpropyl dodecanoate

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: For the purpose of this study the test material was prepared as a 10 % v/v concentration in 0.9 % w/v sodium chloride solution.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks’ Balanced Salt Solution (HBSS) containing the antibiotics Penicillin (100 IU/mL) and Streptomycin (100 µg/mL) and transported to the laboratory on ice packs. The eyes were used within 5 hours of slaughter.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in BCOP holders.
The anterior and posterior chambers of each BCOP holder were filled with fresh complete MEM and plugged. The holders were incubated at 32 ± 1 °C for at least 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride solution

POSITIVE CONTROL USED: ethanol

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 10 min

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Using a syringe to create a whirlpool effect each cornea was rinsed with fresh complete MEM
- POST-EXPOSURE INCUBATION: 120 min in MEM

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others: visual observations

Application of Sodium Fluorescein: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from both chambers (anterior chamber first) was removed. The posterior chamber was refilled with fresh complete MEM and 1 mL of 4 mg/mL sodium fluorescein was applied to the anterior chamber. The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes ± 5 minutes.
Permeability Determinations: After incubation all the medium in the posterior chamber of each holder was decanted using a syringe with a needle attached and thoroughly mixed in tubes, pre-labelled according to holder number, so that a representative sample was obtained for the OD492 determination. 360 µL of medium representing each cornea was applied to a designated well on a 96 well plate. Two wells were designated as blanks remained empty.These were used for blank subtraction purposes. The optical density at 492 nm (OD492) was measured using the Anthos 2001 micro plate reader. A sodium fluorescein calibration curve was performed to determine the linear range of the testing facilities microplate reader.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS 0 - 25 = mild irritant
IVIS 25.1 -55 = moderate irritant
IVIS 55.1 and above = severe irritant

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1st experiment

Value:

1.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

0.9

Positive controls validity:

valid

Remarks:

77.1

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

The in vitro Irritancy scores are summarised as follows:

Treatment	In Vitro Irritancy Score	Classification
Test Material	1.6	Mild Irritant
Negative Control	0.9	Mild Irritant
Positive Control	77.1	Severe Irritant

As it appears in the table, the test material and the negative control scores are relatively similar and therefore were classified identically in this table. Thus, the test item is practically not irritating.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Comparison of the in vitro irritancy scores of the test substance, negative control and positive control showed that the test substance is not an eye irritant.

Executive summary:

The study was performed to assess the ocular irritancy potential of the test substance to isolated bovine cornea. The study followed the procedures of the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recommended protocol for the Bovine Corneal Opacity and Permeability (BCOP) test method. The test material was prepared as a 10 % v/v concentration in 0.9 % w/v sodium chloride solution. 0.75 mL of the test material or control materials were applied directly to the epithelial surface of the appropriate corneas (ones that were found at the beginning of the study to be free of damage) for 10 minutes, followed by a post incubation period of 120 minutes with MEM (without the test substance). The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. Once the opacity and mean permeability (OD490) values have been corrected for background opacity and the negative control permeability OD490 values, the mean opacity and permeability OD490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro score (IVIS) for each treatment group. The in vitro irritancy scores for the test material (1.6) and the negative control (0.9 % sodium chloride solution) (0.9) were relatively similar while the result of the positive control (ethanol) was as expected high (77.1). A substance that induces an IVIS (in vitro score) ≥ 55.1 is defined as a corrosive or severe irritant, therefore the test material was not considered to be a severe skin irritant. Based on the obtained results, the test item is not an eye irritant and no further tests are required.