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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07-07-2017 to 02-03-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: July 2015 ; signature: September 2015

Test material

Constituent 1
Chemical structure
Reference substance name:
dec-1-en-4-yne
EC Number:
813-331-8
Cas Number:
24948-66-1
Molecular formula:
C10H16
IUPAC Name:
dec-1-en-4-yne
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: At 2-8 °C in the refrigerator with nitrogen cover gas
- Other: Slightly yellow

Test animals / tissue source

Species:
other: bovine
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Recognised supplier (documented in the full study report)
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): > 9 months old (typically).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. 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. The corneae were isolated on the same day after delivery of the eyes.
Post-excision, placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics over ice packs.
- Time interval prior to initiating testing: < 24 hours. Corneas were prepared for testing immediately on same day arrival.
- indication of any existing defects or lesions in ocular tissue samples: None. Only corneas free from damage utilised (e.g. presenting defects such as vascularization, pigmentation, opacity and scratches were discarded).
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 μg/mL during transport.

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
- Concentration (if solution): undiluted
Duration of treatment / exposure:
10 minutes at 32 ± 1ºC.
Duration of post- treatment incubation (in vitro):
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. A post-treatment opacity reading was taken and each cornea was visually observed (t130).
Number of animals or in vitro replicates:
Three (3) per test item, or negative or positive controls, respectively.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. Following mounting: the anterior and posterior chambers of each BCOP holder were filled with Incubation medium. The incubation medium consists of MEM, supplemented with 1.1 g sodium bicarbonate, 5 mL L-glutamine, 5 mL penicillin/streptomycin per 500 mL medium (final concentration of 100 units penicillin per mL medium, and 100 μg streptomycin per mL medium). Immediately before starting the test, MEM was supplemented with 1% fetal calf serum (FCS). The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that 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 pre-exposure incubation period, the basal opacity was determined (t0).

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were examined for defects macroscopically (e.g. presenting defects such as vascularization, pigmentation, opacity and scratches) and where necessary discarded. Additionally, only corneas with opacity < 7.0 are discarded, in accordance with the guideline.

NUMBER OF REPLICATES: 3 (Triplicate)

NEGATIVE CONTROL USED: 0.9% w/v Sodium chloride solution

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: 2-ethoxyethanol; 99.0% purity

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 minutes at 32 ± 1ºC.

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: 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 pre-exposure incubation period, the basal opacity was determined (t0).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber. After the test item or control items, respectively, were rinsed off from the application side with saline, fresh complete medium (cMEM) was added into the anterior compartment.

- POST-EXPOSURE INCUBATION: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. A post-treatment opacity reading was taken and each cornea was visually observed (t130).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured through light transmission through the cornea quantitatively using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Any other pertinent visual observations would be recorded.

SCORING SYSTEM: Opacity, Permeability and In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The mean opacity and mean permeability values (OD492) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD492 value). A test item that induces an In Vitro Irritancy Score >/=55.1 is defined as an ocular corrosive or severe irritant. A test item with an IVIS

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean (n=3)
Value:
2.09
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:
OTHER EFFECTS:
- Visible damage on test system: None. The corneas treated with the test item did not cause a relevant increase of the corneal opacity. For corneas treated with the negative control item neither an increase of opacity nor permeability could be observed. The corneas treated with the positive control item was tested undiluted and showed clear opacity and distinctive permeability of the corneae.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test facility was a validated laboratory (information in the public domain) and/or concurrent positive and negative controls were within acceptable limits.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline:
1. 2-ethoxyethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the current historical control data (HCD) mean of for the test facility. This criterion was met (actual mean IVIS = 92.17)
2. sodium chloride 0.9% w/v solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values for bovine corneas treated with the respective negative control of the current historical control data (HCD). This criterion was met (actual mean opacity = 0.00 ; permeability = 0.059 and IVIS = 0.88)
Full details of the HCD is provided in the full study report.

Any other information on results incl. tables

 1. Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD490)

In Vitro Irritancy Score

Post-Incubation - Pre‑Treatment

 

Negative Control

1

0

0.055*

0.83

2

0

0.064*

0.96

3

0

0.057*

0.86

Mean

0.00

0.059*

0.88

Positive Control

4

78.00*

0.894*

91.42

5

75.00*

0.849*

87.74

6

85.00*

0.774*

97.37

Mean

-

-

92.17

Test Item

7

2.00*

0.003*

2.05*

8

2.00*

0.009*

2.14*

9

2.00*

0.004*

2.07*

Mean

-

-

2.09

OD = Optical Density

* = corrected values

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item is not considered to be irritant in the in vitro eye corrosion/irritation test using Bovine Corneal Opacity and Permeability model. The in vitro irritancy score (IVIS) was < 3.0 in the prediction model.
Executive summary:

The study was performed according to OECD TG 437 and EU Method B.47 to assess the eye irritancy potential in accordance with GLP of the test material in isolated bovine corneas. The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The ocular irritancy of the test item was tested through topical application for 10 ± 1 minutes. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (2-ethoxyethanol), was 92.17 (GHS Category 1 eye damage prediction) and was within the historical positive control data range. The test item did not induce ocular irritation through both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score (IVIS) of 2.09 after 10 minutes of treatment. Since the IVIS was < 3.0 the test item was predicated as not irritating to the eye. Under the conditions of this study the test item is not considered to be an irritant or corrosive to the eye in the Bovine Corneal Opacity and Permeability test.