DODECANEDIOIC ACID, 1,12-BIS(2-OCTYLDODECYL) ESTER

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,12-bis(2-octyldodecyl) dodecanedioate

EC Number:

603-333-5

Cas Number:

129423-55-8

Molecular formula:

C52 H102 O4

IUPAC Name:

1,12-bis(2-octyldodecyl) dodecanedioate

Test material form:

liquid

Details on test material:

Lot P-3955

Test animals / tissue source

Species:

cattle

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

positive control applied at 100%
negative control of physiological saline
0.75milliliters used in all cases

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:

Bovine eyes were obtained by this facility from a local abattoir. The eyes were transported in an appropriate container containing Hank's balanced salt solution (HBSS, Sigma Chemical Co., H- 1387). Transportation and storage of the eyes was at approximately 70° F. The eyes were used the day of their harvest and transport.

Upon receipt, all eyes were examined. Magnification was used as needed. Eyes with corneas deemed unacceptable, due to scratches, vascularization. pigmentation, opacity, or for any reason, were discarded.

Accepted corneas were dissected from each eye, using a scalpel. A 2 to 3 millimeter wide piece of sclera was left surrounding the cornea. The corneas were placed in fresh HBSS until the testing began.

Upon test initiation, the iris and the lens were removed and the corneas were placed in the bovine cornea holders. The endothelial surface was applied to the O-ring of the posterior part of the holder. The anterior part of the holder was placed against the epithelial side of the cornea. The two sides of the holder were screwed together. The bovine holders' components were then filled with pre-warmed Eagle's Minimum Essential Media (EMEM, Sigma-Aldrich Lot# 051M8302)
with 1% Fetal Bovine Serum (FBS, American Type Culture Collection) at approximately 32° ±
2° C. The posterior chamber was filled first. The corneas were incubated for approximately one hour, in a 32° ± 2° C water bath. This allowed for the pre-equilibration of the corneas to the external medium.

During this pre-equilibration period, the opacitometer was calibrated, without a cornea, as per the operator's manual. The electrical zero (balance between photocells) was adjusted to "0" with the "bal." knob. The apparatus was set to "75" with a standardized opaque sheet of polyester, placed in the "positive" compartment.

After the pre-equilibration period, media from both of the chambers of the cassettes was aspirated, with the anterior chamber being aspirated first. Each chamber was then refilled with fresh EMEM, with the posterior chamber being filled first. Initial opacity readings were taken and recorded. Corneas with initial readings greater than 003 were not used. Three corneas were assigned to each test and control article. The corneas with the lowest readings were used as the negative controls.
The EMEM was then removed from the anterior chambers.

Opacity Measurement:

The test article was tested at 100%. The positive control (ethanol) and the negative control (physiological saline) were also each tested at 100%.

A volume of 0.75 milliliters of the appropriate article, pre-warmed to approximately 32° ± 2° C, was introduced into the anterior chamber of each of the appropriate cassettes. The anterior chambers were then plugged and turned to a horizontal position. They were then rotated slightly, so that the article uniformly covered each cornea. The cassettes were incubated for 10 minutes at 32° ± 2° C, in a water bath.
After the 10 minute exposure period, the articles were removed and the corneas were washed at least 3 times (or until the wash medium appeared clear). Each wash consisted of approximately 3 milliliters of EMEM being added to the anterior chambers via syringe. The media was removed using a syringe with a small intubation needle.

The anterior chambers of each cassette were then refilled with fresh EMEM via syringe. After refilling, the chambers were again plugged. All corneas were then incubated for 2 hours at approximately 32° ± 2° C, in a water bath.

After this 2 hour incubation period, all compartments were emptied and refilled with fresh EMEM. Each holder was then individually placed in the "positive" compartment of the Opacitometer, while the "negative" compartment was left empty. The glass portion of each holder was dried prior to the reading. The opacity of each cornea was taken and recorded. The corneas were then observed grossly. Any unusual findings, such as opaque spots, were recorded.

Permeability Determination:

After the opacity readings were completed, the medium was removed from the anterior chambers. A syringe with a small intubation needle was used for this procedure. One milliliter of 4 mg/ml fluorescein solution (Sodium Fluorescein, Sigma-Aldrich, Batch# 064K0153 in Phosphate Buffered Saline (DPBS)) was added to each of the anterior chambers.

After the fluorescin solution was added to each anterior compartment, the compartments were plugged. The cassettes were then incubated, in a horizontal position, for 90 minutes at 32° ± 2° C, in a water bath. After the 90 minute incubation, the medium in the posterior chambers was mixed by drawing approximately 1 milliliter gently, up and down three times. A 1 milliliter syringe, with a small intubation needle, was used for this procedure.

A 200 microliter volume of the EMEM was then drawn from the posterior chamber of each cassette. The optical density (OD490) of the EMEM sample was then measured spectrophotometrically, in a microplate reader at 490 nm, using 200 microliters of the fresh EMEM as the blank.

Evaluation of Results:

An In Vitro Score was determined from the opacity and permeability measurements.

Opacity the difference in opacity value of each treated cornea (test article, positive control article and negative control article) was calculated by subtracting the initial, basal opacity reading from the post treatment opacity reading. This value was determined for each cornea.

The average change in opacity for the negative control corneas was calculated. This value was subtracted from the change in opacity of each treated cornea (test article and positive control article) to obtain a corrected opacity.

The mean corrected corneal opacity value was determined from the individual, corrected opacity values of the corneas exposed to the test article and the positive control article.

Permeability the corrected OD490 values (permeability) of the test article and the positive control article treated corneas were calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

The mean corrected permeability value of each treatment group was determined from the individual corrected permeability values of the corneas exposed to the test article and the positive control article.

The In Vitro Score for each individual test article and pos1t1ve control article cornea was determined as follows: In Vitro Score = Corrected Corneal Opacity Value + (15 x Corrected OD490 value). The mean In Vitro Score value for the test article and the positive control article was calculated from the individual In Vitro Score values.
The following classification system was established by Gautheron, et. al. (1992) and refined by Vanparys et. al. (1994). This system is intended to classify articles tested under standard conditions and is intended as a general guide. Results from the test conditions should be compared to known articles tested under the same, or similar, conditions.


3 REPLICATES

NEGATIVE CONTROL USED physiological saline

POSITIVE CONTROL USED 100% ethanol

APPLICATION DOSE AND EXPOSURE TIME 100% for 10 minutes

TREATMENT METHOD: bovine corneal holders

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 with EMEM until clear
- POST-EXPOSURE INCUBATION: 2 hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Each holder was then individually placed in the "positive" compartment of the Opacitometer, while the "negative" compartment was left empty. The glass portion of each holder was dried prior to the reading. The opacity of each cornea was taken and recorded. The corneas were then observed grossly. Any unusual findings, such as opaque spots, were recorded.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) cross referencing to the Draize score

DECISION CRITERIA: Draize table - see below

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

ca. 0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Article        Cornea#		Initial Opacitv	Terminal Opacity	Difference	Corrected Corneal Opacity	Mean Corr. Corneal Opacity
Test                    I		2	7	5	0.3
Test                    2		0	6	6	1.3
Test                    3		0	4	4	-0.7	0.3
+ Control         12		I	43	42	37.3
+ Control         13		0	44	44	39.3
+ Control         14		0	41	41	36.3	37.6
- Control	5	-1	2	3
- Control	7	-2	2	4
- Control	11	-1	6	7 (Average= 4.7)

 

 

Article	Cornea#	Optical Density490	Corrected OD490	Mean Corr. OD490	In Vitro Score	Average In Vitro Score
Test	1	0.013	0.007		0.4
Test	2	0.012	0.006		1.4
Test	3	0.017	0.011	0.008	-0.5	0.4
+ Control	12	1.447	1.441		58.9
+ Control	13	1.638	1.632		63.8
+ Control	14	1.736	1.730	1.601	62.3	61.7
- Control	5	0.007
- Control	7	0.008
- Control	11	0.004 (Average= 0.006)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this test, the sponsor-submitted product at I 00%, elicited an average in vitro score of 0.4 and the positive control article, ethanol, at 100%, elicited an average in vitro score of 61.7. Therefore it is concluded that the test article, at 100%, is non-irritating and would be expected to elicit a Draize in vivo score approaching 0, on a scale of 0 to 110.

Executive summary:

Product should not be classified according to CLP criteria for eye irritation