Amines, rosin

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

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)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006

Version / remarks:

The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM).

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: In Vitro Techniques in Toxicology Database (INVITTOX) study plan 127. Bovine Opacity and Permeability (BCOP) Assay, 2006

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Amines, rosin

EC Number:

263-139-8

EC Name:

Amines, rosin

Cas Number:

61790-47-4

Molecular formula:

C19H31N1

IUPAC Name:

1-[(1R,4aR,10aS)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl]methanamine; 1-[1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl]methanamine

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rosin amine 90
- Expiration date of the lot/batch: 31 March 2016
- Purity test date: No correction factor required
- pH (1% in water, indicative range) 7.2 – 8.5 (determined by WIL Research Europe)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No correction was made for the purity/composition of the test item. The test item was tested neat.

OTHER SPECIFICS:
Negative control:
A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

Positive control:
10% (w/v) Benzalkonium Chloride (Merck KGaA, Darmstadt, Germany) [CAS Number 63449-41-2] solution prepared in physiological saline.

Test animals / tissue source

Species:

other: Bovine eyes

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the ey
- Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
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.

PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Eagle’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
- Following the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations will be performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany).
- The opacity of each cornea will be read against a cMEM filled chamber, and the initial opacity reading thus determined will be 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 system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µl of either the test substance, negative or positive control was introduced onto the epithelium of the cornea.

Duration of treatment / exposure:

10 ± 1 minutes at 32 ± 1°C

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

Not applicable

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. 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.
- After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations was 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.
- After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’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.
- Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck Darmstadt, Germany) was evaluated.

SCORING SYSTEM:
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
 - In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
 - Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

TOOL USED TO ASSESS SCORE: opacitometer

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Value:

29.9

Negative controls validity:

valid

Remarks:

-0.6

Positive controls validity:

valid

Remarks:

97.1

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Remarks:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Value:

55.3

Negative controls validity:

valid

Remarks:

-0.6

Positive controls validity:

valid

Remarks:

144.1

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
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 (10% (w/v) Benzalkonium Chloride) was 144 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.

ACCEPTANCE OF RESULTS: Yes
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean = Yes: the mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 144 and was within two standard deviations of the current historical positive control mean
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range = Yes: 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

- Historical control data can be found in the 'Attached background information' below.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Rosin Amine 90 induced serious eye damage through both endpoints: Corneal Opacity and Permeability, resulting in a mean in vitro irritancy score of 55 after 10 minutes of treatment.

Executive summary:

The eye hazard potential of the test substance, Rosin Amine 90, was evaluated using the Bovine Corneal Opacity and Permeability test (BCOP test), in a procedure compatible with OECD 437, EC Commission Regulation No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.47 'Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants, OTWG: The Bovine Corneal Opacity and Permeability (BCOP) Test Method (March 2006), In Vitro Techniques in Toxicology Database study plan 127: Bovine Opacity and Permeability (BCOP) Assay (2006) and Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy (Gautheron, P. et al., 1992).

 

The test substance's potential to cause eye damage was tested through topical application of 750 µl directly to the top of bovine corneas for 10 minutes. The same volume of positive and negative controls were also tested. The negative control was physiological saline, employed in order to detect non-specific change in the test system and to provide a baseline for the assay endpoints. The positive control used was 10% (w/v) Benzalkonium Chloride solution, prepared in physiological saline.

 

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 (10% (w/v) Benzalkonium Chloride) was 144 and was within two standard deviations of the current historical positive control mean. These results met the acceptability criteria, and therefore can be concluded that the test conditions were adequate and that the test system functioned properly.

 

Rosin Amine 90 induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 55 after 10 minutes of treatment. Therefore, it can be concluded that Rosin Amine 90 induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report and should be classified category 1 according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.