Isotridecyl isononanoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 November 2017 - 10 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Isotridecyl isononanoate

EC Number:

255-673-5

EC Name:

Isotridecyl isononanoate

Cas Number:

42131-27-1

Molecular formula:

C22H44O2

IUPAC Name:

Isotridecyl isononanoate

Specific details on test material used for the study:

Identification: Isotridecyl isononanoate
CAS No.: 42131-27-1
Batch: 0000807803
Purity: 100%
Appearance: Colourless to yellowish* liquid
Expiry Date: 06 April 2018
Storage Conditions: At room temperature
Stability in Solvent: Stable in water (not quantified)
Purpose of Use: Industrial chemical

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Collection of Bovine Eyes
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.

Test system

Vehicle:

Hank's balanced salt solution

Controls:

yes

Amount / concentration applied:

The test item was tested undiluted.
The anterior compartment received the test item or the negative or positive controls at a volume of 0.75 mL on the surface of the corneae, respectively.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Details on study design:

Preparation of Corneae
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 according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. 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 incubation medium. 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 end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and for the negative and positive controls.

Exposure of the Corneae to the Test Groups
The corneae were distributed as follows:
Group 1: Negative Control - 3 Corneae
Group 2: Positive Control - 3 Corneae
Group 3: Test item - 3 Corneae

The anterior compartment received the test item or the negative or positive controls at a volume of 0.75 mL on the surface of the corneae, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted ten minutes.
After the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added into the anterior compartment. Then the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130).
In the second step of the assay, permeability of the corneae was determined.

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130).

Permeability Determination
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Data Evaluation
Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0).
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

3.8.3 In vitro Score (IVIS) Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS ≤3 : No Category
IVIS > 3; ≤55 : No prediction can be made
IVIS >55 : Category 1

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

0.26

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

1.26

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

0.89

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

0.8

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Results after 10 Minutes Treatment Time

Test Group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score
		Mean		Mean
Negative Control	0	0.00	0.054	0.064	0.81	0.96	No Category
	0		0.062		0.93
	0		0.075		1.13
Positive Control	71.00*		1.123*		87.85	88.18	Category 1
	67.00*		1.383*		87.75
	62.00*		1.796*		88.95
Test Item	0.00*		0.017*		0.26	0.80	No Category
	1.00*		0.017*		1.26
	1.00*		-0.008*		0.89

*corrected values

Thisin vitrostudy was performed to assess thecorneal damage potentialofIsotridecyl isononanoateby means of the BCOP assay usingfresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t₀), the neat test item Isotridecyl isononanoate, the positive, and the negative controls were applied to the different corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneae. Further, the corneae wereincubated for another 120 minutes at 32 ± 1 °C in incubation medium, andopacity was measured for 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 (saline) neither an increase of opacity nor permeability of the corneae could be observed (meanIVIS= 0.96).

The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (meanIVIS =88.18) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

The test item Isotridecyl isononanoate was tested undiluted. Relative to the negative control, the test item Isotridecyl isononanoate did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 0.80 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not categorized.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, Isotridecyl isononanoate is not categorized (GHS).