2-ethylhexyl 4-(dimethylamino)benzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

2013

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:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 4-(dimethylamino)benzoate

EC Number:

244-289-3

EC Name:

2-ethylhexyl 4-(dimethylamino)benzoate

Cas Number:

21245-02-3

Molecular formula:

C17H27NO2

IUPAC Name:

2-ethylhexyl 4-(dimethylamino)benzoate

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Characteristics of donor animals: Cattle used were typically 12 to 60 months old.
- Storage, temperature and transport conditions of ocular tissue: Eyes were transported to the test facility over ice packs in supplemented Hanks’ Balanced Salt Solution (HBSS).
- Time interval prior to initiating testing: Eyes were transported to the test facility on the same day of slaughter. The corneas were prepared immediately on arrival.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: Yes; eyes were placed in HBSS supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL).

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

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes after the treatment with the test material
90 minutes with Sodium fluorecein

Number of animals or in vitro replicates:

3 replicates for each condition (test material, negative control and positive control)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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 in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 °C for 60 minutes.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

TREATMENT METHOD
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test material or control materials were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 10 minutes.
At the end of the exposure period the test material and control materials were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 °C for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The corneal opacity readings were taken using a calibrated opacitometer. The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: Following the final opacity measurement, medium was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes to measure permeability. After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader. The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
- Others: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10 % neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.

ACCEPTABILITY OF THE ASSAY
For an acceptable test the following control criteria should be achieved:
-The test was acceptable if the positive control produced an IVIS which fell within two standard deviations of the historical mean collated during 2014 for the testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 29.6 to 52.0.
-The test was acceptable if the negative control produced an IVIS which is less than or equal to the upper limit for background opacity and permeability values during 2014 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be = 2.9 and for permeability = 0.103.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

A summary of the results can be seen in Table 1. The corneas treated with the test material and negative control material were clear post treatment and post incubation. The corneas treated with the positive control material were cloudy post treatment and post incubation

ACCEPTANCE OF RESULTS
The positive control IVIS score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of =2.9 and permeability =0.103. The negative control acceptance criteria were therefore satisfied.

Any other information on results incl. tables

Table 1: Summary of opacity, permeability and in vitro irritancy scores

Treatment	Mean Opacity	Mean Permeability	Mean In Vitro Irritancy Score
Negative control	0.3	0.008	0.5
Positive control	32.3	1.206	50.4
Test material	0.2	0.021	0.5

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Not eye irritating

Conclusions:

Under the conditions of this study, the test material had an IVIS score of 0.5 and was therefore determined to require no classification.

Executive summary:

The potential of the test material to cause serious eye damage was determined in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions using the Bovine Corneal Opacity and Permeability (BCOP) assay.

In this test method, damage by the test material is assessed by quantitative measurements of changes in corneal opacity and permeability. The undiluted test material was applied to the corneas for 10 minutes followed by an incubation period of 120 minutes. Negative and positive controls, sodium chloride and ethanol respectively, were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The IVIS scores for the test, negative and positive control materials were 0.5, 0.5 and 50.4, respectively. The corneas treated with the test material and negative control material were clear post treatment and post incubation. The corneas treated with the positive control material were cloudy post treatment and post incubation

The positive and negative controls met the acceptability criteria of the study and the so the test is considered to be valid.

Under the conditions of this study, the test material had an IVIS score of 0.5 and was therefore determined to require no classification.