Reaction mass of trimethylolpropane triacrylate and hexamethyleneimine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 March 2018 to 28 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 (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of trimethylolpropane triacrylate and hexamethyleneimine

EC Number:

946-043-7

IUPAC Name:

Reaction mass of trimethylolpropane triacrylate and hexamethyleneimine

Test material form:

liquid

Specific details on test material used for the study:

Appearance: Colourless to pale yellow liquid
Purity/Composition: 100% (UVCB)
Test item storage: At room temperature

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Specification
Corneas from bovine eyes were obtained from a local abattoir. The eyes were removed after slaughter, completely immersed in physiological saline in a suitably sized container and transported on the same day to the testing facility.

Assessment on Arrival
On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

Excision and Preparation of Corneas
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). 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 anterior half of the holder was positioned on top of the cornea and tightened with screws. 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± 1°C.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The negative control substance was physiological saline, 750 µL, supplied by Eurovet Animal Health, Bladel, The Netherlands
The positive control substance was ethanol, 750 µL
The test article was administered without dilution.

Duration of treatment / exposure:

120 ± 10 minutes at 32±1°C.

Duration of post- treatment incubation (in vitro):

Corneas were incubated in a horizontal position for 10±1 minutes at 32±1°C

Number of animals or in vitro replicates:

3

Details on study design:

In total 2 experiments were performed. 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 (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). 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 anterior half of the holder was positioned on top of the cornea and tightened with screws. 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 ± 1°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.

Test Item Preparation:
No correction was made for the purity/composition of the test item. The test item was tested neat.

Treatment of Corneas and Opacity Measurements:
The medium from the anterior compartment was removed and 750 µl 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 ± 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) 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°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

Opacity Measurement:
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to:

Opacity=(I_0/I-0.9894)/0.0251

With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.

The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Application of Sodium Fluorescein:
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) 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 (Sigma-Aldrich Chemie GmbH, Germany)/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°C.

Permeability Determinations:
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

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment 1 Mean Value

Value:

ca. 2.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean Opacity 2.2

Positive controls validity:

valid

Remarks:

Mean Opacity 21

Remarks on result:

not determinable

Results 2

Irritation parameter:

other: Corneal Permeability

Run / experiment:

Experiment 1, Mean Value

Value:

ca. -0.046

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean Permeability 0.061

Positive controls validity:

valid

Remarks:

Mean Permeability 2.903

Remarks on result:

not determinable

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment 2 Mean Value

Value:

ca. 3.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean opacity 2.1

Positive controls validity:

valid

Remarks:

Mean opacity 23

Remarks on result:

not determinable

Results 4

Irritation parameter:

other: Corneal Permeability

Run / experiment:

Experiment 2, Mean Value

Value:

ca. 0.089

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean Permeability 0.010

Positive controls validity:

valid

Remarks:

Mean Permeability 2.768

Remarks on result:

not determinable

Other effects / acceptance of results:

RESULTS:
Photomer 4250 was tested neat.

Table 1 of Appendix 1 summarizes the opacity, permeability and in vitro irritancy scores of Photomer 4250 and the controls. The opacity, permeability and in vitro scores of the individual corneas are shown in Table 2 - 5.

In the first experiment, the individual in vitro irritancy scores for the negative controls ranged from 2.5 to 3.8. The individual positive control in vitro irritancy scores ranged from 60 to 69 (Appendix 2,
Table 5). The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with Photomer 4250 showed opacity values ranging from 1.0 to 4.1 and permeability values ranging from -0.059 to -0.024. The corneas were translucent after the 10 minutes of treatment with Photomer 4250. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.2 to 3.8 after 10 minutes of treatment with Photomer 4250.
Since the results were spread over 2 categories (3.8, 2.7 and 0.2), the test was inconclusive and a repeat experiment was performed.

In the second test, the individual in vitro irritancy scores for the negative controls ranged from 2.0 to 2.6. The individual positive control in vitro irritancy scores ranged from 55 to 75 (Appendix 2, Table 5). The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with Photomer 4250 showed opacity values ranging from 2.7 to 4.1 and permeability values ranging from -0.018 to 0.263. The corneas were translucent after the 10 minutes of treatment with Photomer 4250. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 3.4 to 6.7 after 10 minutes of treatment with Photomer 4250.

DISCUSSION:
The mean 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 64 in both experiments and within two standard deviations of the current historical positive control mean (Appendix 3, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

In the first experiment, the mean in vitro irritancy score was 2.2 after 4 hours of treatment with the test item. The results were spread over 2 categories (3.8, 2.7 and 0.2 respectively).

In the second experiment, Photomer 4250 induced ocular irritation through one endpoint, resulting in a mean in vitro irritancy score of 4.8 after 10 minutes of treatment.

Over the two assays, Photomer 4250 had a mean in vitro irritancy score of 3.5. Furthermore, Photomer 4250 induced an IVIS > 3 ≤ 55 in 4 out of 6 cornea’s, therefore no prediction on the classification can be made.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, since Photomer 4250 induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Photomer 4250 as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas.  The eye damage of Photomer 4250 was tested through topical application for 10 minutes.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 17C13003 of Photomer 4250 was a colourless to pale yellow liquid.  The test item was applied as it is (750 µL) directly on top of the corneas.

In the first experiment, the mean 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 64 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.  

The mean in vitro irritancy score was 2.2 after 4 hours of treatment with the test item.

Since the results were spread over 2 categories (3.8, 2.7 and 0.2 respectively), the test was inconclusive and a repeat experiment was performed.

In the second experiment, 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 64 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.  

Photomer 4250 induced ocular irritation, resulting in a mean in vitro irritancy score of 4.8 after 10 minutes of treatment.

Over the two assays, Photomer 4250 had a mean in vitro irritancy score of 3.5. Furthermore, Photomer 4250 induced an IVIS > 3 ≤ 55 in 4 out of 6 cornea’s, therefore no prediction on the classification can be made.

In conclusion, since Photomer 4250 induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.