Reaction products of Soya and Linseed Oil Fatty Acids with Bisphenol A diglycidylether

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 May 2018 to 15 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

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)

Version / remarks:

adopted October 09, 2017).

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

EC Number:

216-823-5

EC Name:

2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Cas Number:

1675-54-3

Molecular formula:

C21H24O4

IUPAC Name:

2,2'-[propane-2,2-diylbis(4,1-phenyleneoxymethylene)]dioxirane

Constituent 2

Reference substance name:

Fatty acids, linseed-oil, polymers with bisphenol A, epichlorohydrin and soya fatty acids

EC Number:

613-885-9

Cas Number:

66070-79-9

Molecular formula:

(C15 H16 O2 . C3 H5 Cl O . Unspecified . Unspecified)x

IUPAC Name:

Fatty acids, linseed-oil, polymers with bisphenol A, epichlorohydrin and soya fatty acids

Test material form:

liquid: viscous

Details on test material:

Clear colourless viscous liquid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes from young cattle were obtained from a local slaughterhouse where the eyes were excised by a slaughterhouse employee as
soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Amount Applied:
0.75 ml of test item, positive control, or negative control

Duration of treatment / exposure:

10 +/- 1 minutes

Duration of post- treatment incubation (in vitro):

120 +/- 10 minutes

Number of animals or in vitro replicates:

3

Details on study design:

Preparation of Corneas
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum . The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder.. The compartments of the corneal holder were filled with cMEM of 32 +/- 1C. The corneas were incubated for the minimum of 1 hour at 32 +/- degrees C.

Cornea Selection and Opacity Reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Treatment of Corneas and Opacity Measurements
The medium from the anterior compartment was removed and 750 ul of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 +/- 1 minutes at 32 +/- degrees C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 +/- 10 minutes at 32 +/- 1 degrees C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1 degrees C.

After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean in vitro irritancy score of -1.4

Value:

> -2.2 - < -0.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Treatment	Mean Opacity^1	Mean Permeability^1	Mean In Vitro Irritation Score^1, 2
Negative control	1.8	-0.016	1.6
Positive control (Ethanol)	16	1.625	40
Soya/Linseed Oil Fatty Acid-BADGE reaction product	-1.7	0.022	-1.4

1   Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

2   In vitroirritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since Soya/Linseed Oil Fatty Acid-BADGE reaction product induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Soya/Linseed Oil Fatty Acid-BADGE reaction product as measured by its ability to induce opacity and increase

permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeabilitytest (BCOP test).

The study procedures were based on the most recent OECD guideline 437. The test item was applied as it is (750 μL) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on

the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 40 and was within two standard deviations of the current historical positive control mean. It was

therefore concluded that the test conditions were adequate and that the test system functioned properly.

Soya/Linseed Oil Fatty Acid-BADGE reaction product did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.4 after 10 minutes of

treatment.

In conclusion, since Soya/Linseed Oil Fatty Acid-BADGE reaction product induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.