2,2-dimethyl-1,3-propanediyl dioleate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Nov 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

2,2-dimethyl-1,3-propanediyl dioleate

EC Number:

255-713-1

EC Name:

2,2-dimethyl-1,3-propanediyl dioleate

Cas Number:

42222-50-4

Molecular formula:

C41H76O4

IUPAC Name:

2,2-dimethyl-1,3-propanediyl dioleate

Details on test material:

- Name of test material (as cited in study report): 2,2-dimethyl-1,3-propanediyl dioleate
- Physical state: yellowish liquid
- Analytical purity: 100%
- Lot/batch No.: OE 10124A
- Expiration date of the lot/batch: 2013-01-24
- 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.
- Type of cornea holder used: supplied by LAB Research, Veszprem, Hungary
- 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: 1.36-1.72 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

Observation period (in vivo):

not applicable, post-exposure incubation period: 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 vivo

Results

Results 1

Irritation parameter:

other: opacity

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 10 min + 2 h

Score:

0.32

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Test substance

Results 2

Irritation parameter:

other: permeability

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 10 min + 2 h

Score:

0.069

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Test substance

Results 3

Irritation parameter:

other: IVIS

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 10 min + 2 h

Score:

0.747

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Test substance

Results 4

Irritation parameter:

other: opacity

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 10 min + 2 h

Score:

128.418

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Positive control

Results 5

Irritation parameter:

other: permeability

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 10 min + 2 h

Score:

1.907

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Positive control

Results 6

Irritation parameter:

other: IVIS

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 10 min + 2 h

Score:

156.148

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Positive control

Results 7

Irritation parameter:

other: opacity

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 10 min + 2 h

Score:

0.213

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Negative control

Results 8

Irritation parameter:

other: permeability

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 10 min + 2 h

Score:

0.044

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Negative control

Results 9

Irritation parameter:

other: IVIS

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 10 min + 2 h

Score:

0.878

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Negative control

Results 10

Irritation parameter:

other: opacity

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 10 min + 2 h

Score:

0.261

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Solvent control

Results 11

Irritation parameter:

other: permeability

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 10 min + 2 h

Score:

0.023

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Solvent control

Results 12

Irritation parameter:

other: IVIS

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 10 min + 2 h

Score:

0.604

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Solvent control

Irritant / corrosive response data:

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	1.4263 1.6413 1.7231	1.7116 1.8863 2.1533	0.2853 0.2449 0.4302	0.3204	0.0978
Solvent control (olive oil)	1 2 3	1.4511 1.4116 1.6474	1.7422 1.6058 1.9463	0.2911 0.1943 0.2989	0.2614	0.0583
Negative control (0.9% NaCl)	1 2 3	1.5226 1.5286 1.4914	1.8932 1.7669 1.5219	0.3706 0.2382 0.0305	0.2131	0.1714
Positive control (10% NaOH in 0.9% NaCl)	1 2 3	1.5108 1.6361 1.3555	171.475 92.215 126.067	169.964 90.579 124.711	128.418	39.8222

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	0.0083 0.0160 0.0169	5 5 5	0.0415 0.0800 0.0845	0.0687	0.0236
Solvent control (olive oil)	1 2 3	0.0045 0.0046 0.0046	5 5 5	0.0225 0.0230 0.0230	0.0228	0.0003
Negative control (0.9% NaCl)	1 2 3	0.0101 0.0106 0.0059	5 5 5	0.0505 0.0530 0.0295	0.0443	0.0129
Positive control (10% NaOH in 0.9% NaCl)	1 2 3	0.3771 0.3820 0.3852	5 5 5	1.8855 1.9100 1.9260	1.9072	0.0204

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)	0.304 0.842 1.094	0.747	54
Solvent control (olive oil)	0.629 0.539 0.644	0.604	9.4
Negative control (0.9% NaCl)	1.128 1.033 0.473	0.878	40.3
Positive control (10% NaOH in 0.9% NaCl)	197.369 118.351 152.723	156.148	25.3

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	0.878
IVIS of positive control (10% NaOH)	103.3-276.1	156.148

Table 5. IVIS - Historical range of controls

Parameter	IVIS (Negative control)	IVIS (solvent control)	IVIS (Positive control)
Substance	0.9% NaCl	olive oil	10% NaCl
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

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered as severe eye irritant (Serious eye damage Category 1/R41) based on a positive result in the Bovine Corneal Opacity and Permeability (BCOP) test. Negative in-vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant (Category 2/R36) and shall therefore be subject to further evaluation.