Fatty acids, C8-10-(even numberd), Esters with pentaerythritol and adipic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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 Ocular Corrosives and Severe Irritants)

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C8-10, mixed esters with adipic acid and trimethylolpropane

EC Number:

306-085-3

EC Name:

Fatty acids, C8-10, mixed esters with adipic acid and trimethylolpropane

Cas Number:

95912-89-3

IUPAC Name:

95912-89-3

Details on test material:

- Name of test material (as cited in study report): Fatty acids, C8-10, mixed esters with adipic acid and trimethylolpropane
- Physical state: yellowish brown liquid
- Analytical purity: 100%
- Lot/batch No.: OE 10428
- Expiration date of the lot/batch: 2013-04-28
- Storage condition of test material: in a closed vessel at room temperature (20 ± 5 °C)

Test animals / tissue source

Species:

other: cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used to classify substances as “ocular corrosives and severe irritants”. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in isolated bovine corneas. The opacity and permeability assessments of the corneas are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Müller Fleisch GmbH, Birkenfeld, Germany
- Donor animals: 12-60 months
- Transport medium and temperature conditions: Hank’s balanced salt solution (HBSS) supplemented with 0.01% [v/v] penicillin/streptomycin

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: corneas were excised with a scalpel and cut from the globe with a 2 to 3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder, in which pre-warmed complete Minimum Essential Medium (cMEM) without phenol red was filled.
- Description of the cornea holder: the cornea holders consisted of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea.
- Test medium and temperature conditions used in the cornea holder: complete Minimum Essential Medium (cMEM) with and without phenol red, supplemented with foetal bovine serum, L-glutamine, sodium bicarbonate and penicillin/streptomycin; prior to use: pre-warmed to 32 ± 1 °C
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: free of macroscopic defects, initial opacity< 7 (range: 1.35-2.25 for all corneas)

DETERMINATION OF THE INITIAL OPACITY
- Method: corneal opacity was determined by the amount of light transmission through the cornea using a spectral photometer.
- Specification of the device: Spectral photometer Specord 205, Analytik Jena, Germany

Test system

Vehicle:

other: olive oil

Controls:

other: number of corneas for the solvent control: 3; number of corneas for the negative control: 3; number of corneas for the positive control: 3

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied in the test: 750 µL
- Concentration (if solution): 10%

SOLVENT CONTROL
- Substance: olive oil
- Amount(s) applied in the test: 750 µL

NEGATIVE CONTROL
- Substance: 0.9% sodium chloride
- Amount(s) applied in the test: 750 µL

POSITIVE SUBSTANCE
- Substance: sodium hydroxide
- Concentration (if solution): 10% in 0.9% sodium chloride
- Amount(s) applied in the test: 750 µL

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

number of corneas for the test item: 3

Details on study design:

TEST CONDITIONS
- Short description of the method used: closed-chamber method
The respective substance (negative control, positive control, solvent control or the test item solution) was applied by pipetting 750 µL of the appropriate solution through the refill hole in the holder on the cornea. The test item was applied to the epithelium of the cornea in such a manner that as much as possible of the cornea was covered with the test item solution. Each cornea was exposed for a period of 10 min at 32 °C.

POST-EXPOSURE TREATMENT
- Removal of the test substance: first, corneas were rinsed with cMEM containing phenol red followed by a final rinsing with cMEM without phenol red. Afterwards, the anterior chamber was filled with cMEM (without phenol red), and the corneas were stored for an additional 2 h at 32 °C (post-incubation).
- Medium for washing the corneas: cMEM containing phenol red
- Medium for final rinsing: cMEM without phenol red

DETERMINATION OF THE FINAL OPACITY
- Method: corneal opacity was determined by the amount of light transmission through the cornea using a spectral photometer.
- Time of determination: after the post-exposure treatment, the cMEM without phenol red was renewed in both chambers and final opacity was recorded at 570 nm.
- Specification of the device: Spectral photometer Specord 205, Analytik Jena, Germany

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: the cMEM without phenol red was removed from the anterior chamber and fluorescein solution was added. The chambers were closed again and incubated for the respective period. After incubation, the content of the posterior chamber was thoroughly mixed, and the concentration of fluorescein solution in this chamber was measured at 490 nm using a spectral photometer.
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (4 mg/mL)
- Incubation time: 90 ± 5 min at ca. 32 ± 1 °C
- Specification of the spectrophotometer: Spectral photometer Specord 205, Analytik Jena, Germany

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

10 min of exposure

Value:

0.217

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Based on the measurements for opacity and permeability, an In Vitro Irritancy Score (IVIS) ≤ 55 was calculated after treatment with the test substance. The positive and negative controls showed the expected results.

Any other information on results incl. tables

Table 1. Opacity scores after 10 min exposure and 2 h post-treatment incubation period

Treatment group	Replicate	Initial opacity	Final opacity	Opacity difference	Mean	SD
Test substance (10% in olive oil)	1 2 3	2.2496 1.6110 1.7183	2.2341 1.8754 2.2090	-0.0155 0.2644 0.4907	0.2465	0.2536
Solvent control (olive oil)	1 2 3	1.4531 1.4904 1.4178	1.5136 1.6300 1.6527	0.0604 0.1397 0.2350	0.1450	0.0874
Negative control (0.9% NaCl)	1 2 3	1.5646 1.5449 1.5115	1.8557 1.8963 1.6734	0.2911 0.3514 0.1619	0.2681	0.0968
Positive control (10% NaOH in 0.9% NaCl)	1 2 3	1.6630 1.3465 1.5985	174.381 174.864 185.353	172.718 173.517 183.755	176.663	6.155

SD = standard deviation

Table 2. Permeability score after 10 min exposure and 2 h post-treatment incubation period

Treatment group	Replicate	OD490	Correction factor	Corrected OD490	Mean	SD
Test substance (10% in olive oil)	1 2 3	0.0048 -0.0012 -0.0009	5 5 5	0.0240 -0.0060 -0.0045	0.0045	0.0169
Solvent control (olive oil)	1 2 3	-0.0018 -0.0003 0.0002	5 5 5	-0.0090 -0.0015 0.0010	-0.0032	0.0052
Negative control (0.9% NaCl)	1 2 3	0.0037 0.0039 0.0088	5 5 5	0.0185 0.0195 0.0440	0.0273	0.0144
Positive control (10% NaOH in 0.9% NaCl)	1 2 3	0.3579 0.3795 0.4826	5 5 5	1.7895 1.8975 2.4130	2.0333	0.3332

SD = standard deviation

Table 3. In vitro irritancy score (IVIS) after 10 min exposure and 2 h post-treatment incubation period

Treatment group	IVIS
	per cornea	per group
		mean	RSD [%]
Test substance (10% in olive oil)	0.569 0.644 0.822	0.217	19.2
Solvent control (olive oil)	-0.075 0.117 0.250	0.098	166.5
Negative control (0.9% NaCl)	0.248 0.077 0.326	0.678	58.6
Positive control (10% NaOH in 0.9% NaCl)	198.883 201.302 219.272	206.486	5.4

RSD = relative standard deviation

Table 4. Validity criteria (according to OECD 437)

Parameter	Criterion	Mean value (present study)
IVIS of negative control (0.9% NaCl)	0-3	0.678
IVIS of positive control (10% NaOH)	103.3-276.1	206.486

Table 5. IVIS - Historical range of controls

Parameter	IVIS (Negative control)	IVIS (solvent control)	IVIS (Positive control)
Substance	0.9% NaCl	olive oil	10% NaOH
Mean	0.456	0.456	189.7
Standard deviation	0.773	0.352	43.2
Range (min-max)	-1.420-2.592	0.123-1.072	107.7-333.3
Range (Validity)	0-3	0-3	103.3-276.1

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive (Eye Irrit. 2 or not classified) according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the test, the test substance was shown to have no corrosive potential in the Bovine corneal opacity and permeability (BCOP) test prediction model. The result does not allow for the non-classification or classification as irritant of the test substance and therefore further evaluation and/or data generation is required.