Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-05-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

EC Number:

919-489-5

Molecular formula:

C13H23N

IUPAC Name:

Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir
- Number of corneae: 9
- Characteristics of donor animals: 9 month old
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported to the laboratory in HBSS at ambient temperature. The corneae were isolated on the same day after delivery of the eyes.
- Time interval prior to initiating testing: The corneae were directly used in the BCOP test on the same day.
- indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL

Duration of treatment / exposure:

10 min

Duration of post- treatment incubation (in vitro):

After the test item or control items were rinsed off from the application side with saline, the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading.

Number of animals or in vitro replicates:

3 corneas were used per group (test item, negative and positive control)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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. Each isolated cornea was mounted in a specially designed cornea holder that consists of anterior and posterior compartments, which interlace with the epithelial and endothelial sides of the cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
The basal opacity of all corneae was recorded. Each corneae with a value of the basal opacity > 7 was discarded.

NUMBER OF REPLICATES
Sets of three corneae were used for treatment with the test item and the negative and positive controls.

NEGATIVE CONTROL USED
Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED
2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL of undiluted substance, 10 min exposure time

POST-INCUBATION PERIOD: yes, two hours after rinsing

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: no number determined. The test item was rinsed off.

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Changes in the light transmission passing through the corneae were determined using an opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometry (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) was used.

DECISION CRITERIA:
IVIS ≤ 3: No Category (according to GHS)
IVIS >3 ≤ 55: No prediction can be made
IVIS > 55: Serious eye damage according to CLP/EPA/GHS (Cat 1)

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

Remarks:

0.69

Positive controls validity:

valid

Remarks:

79.37

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not reported.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.69).
- Acceptance criteria met for positive control: Yes. The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 79.37)
- Range of historical values if different from the ones specified in the test guideline: Negative control: 0.87 – 1.42; positive control: 56.25 – 80.65

Any other information on results incl. tables

Results after 10 min incubation time

Test group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy Score
		Mean		Mean
Negative control	-1	-0.33	0.082	0.068	0.23	0.69	Not categorized
	0		0.065		0.98
	0		0.058		0.87
Positive control	50.33		1.348		70.55	79.37	Category 1
	67.33		1.395		88.25
	53.33		1.731		79.29
Test item	0.33		-0.008		0.21	0.50	Not categorized
	0.33		-0.013		0.13
	1.33		-0.011		1.16

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this in vitro assay, the test substance is considered as not requiring classification for eye irritation or serious eye damage.

Executive summary:

The in vitro study was performed to assess the corneal damage potential of the test substance by means of the BCOP assay using fresh bovine corneae. The study was conducted according to OECD 437. After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t130). 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. With the negative control (0.9 % (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 0.50. According to OECD 437 the test item is considered as not requiring classification for eye irritation or serious eye damage.