Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31/7/2017 - 13/12/2017

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

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)

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)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid

IUPAC Name:

Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid

Test material form:

solid: bulk

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: 0001230562
- Expiration date of the lot/batch: 08 June 2019
- Purity test date: 100 % UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark.
- Stability under test conditions: Assumed to be stable.
- Solubility and stability of the test substance in the solvent/vehicle: Assumed to be stable, dissolved test item is assumed to be homogeneous in solution.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not specified.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: 20% w/v solution.
- Final preparation of a solid: 20% w/v solution in sodium chloride 0.9% w/v.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : In solution.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir. The eyes were excised after slaughter and placed in Hanks’ Balanced Salt Solution supplemented with penicillin at 100 IU/mL and streptomycin at 100 µg/mL. They were transported to the test facility on ice packs on the same day and the corneas were prepared immediately on arrival.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75mL applied per prepared cornea.
- Concentration (if solution): 20% w/v test item solution in sodium chloride 0.9% w/v.

Duration of treatment / exposure:

240 minutes.

Duration of post- treatment incubation (in vitro):

90 minutes (for permeability assay).

Number of animals or in vitro replicates:

3 per treatment (Test item, Negative control, Positive control).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas were incubated in Eagle’s Minimum Essential Medium (EMEM) without phenol red at 32 ± 1 ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

NUMBER OF REPLICATES
3 per treatment type (Test item, negative control, positive control).

NEGATIVE CONTROL USED
0.9% w/v sodium chloride

POSITIVE CONTROL USED
20% w/v Imidazole solution in 0.9% w/v sodium chloride

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL of 20% w/v test item solution in sodium chloride 0.9% w/v applied to cornea at 32 +- 1 ºC for 240 minutes.

TREATMENT METHOD: BCOP holder with anterior and posterior chambers. EMES in posterior chamber and test solution in anterior chamber.

POST-INCUBATION PERIOD: Yes, for permeability determination the corneas were incubated with a 5 mg/mL sodium fluorescein solution at 32 ± 1 ºC for 90 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Three washes with fresh complete EMEM containing phenol red and then a final final rinse with complete EMEM without phenol red.

POST-EXPOSURE INCUBATION: for permeability determination the corneas were incubated with a 5 mg/mL sodium fluorescein solution at 32 ± 1 ºC for 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A pre-treatment and a post-treatment opacity reading was taken for each cornea using a calibrated opacitometer and each cornea was visually observed.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a Labtech LT-4500 microplate reader (quantitative viability analysis) at 492 nm (without a reference filter).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) .

DECISION CRITERIA: Decision criteria as indicated in the testing guidelines.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item

Value:

142.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative Control

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive control

Value:

116.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: All eyes were macroscopically examined before and after dissection for damage and prepared corneas were visually examined for defects.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes .
- Range of historical values if different from the ones specified in the test guideline: No, values fall within study acceptance criteria.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Eye damage as a result from exposure to test item (In vitro irritancy score of 142.5) resulted in a classification of "Category 1. UN GHS or EU CLP Causes serious eye damage".

Executive summary:

The study investigates the potential for Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid to cause in vitro eye irritation. This study was performed using The Bovine Corneal Opacity and Permeability (BCOP) assay according to the OECD Guideline for the Testing of Chemicals No. 437 “Bovine Corneal Opacity and Permeability Assay” and the EU Method B.47 of Commission Regulation (EC) No. 440/2008. The study is GLP compliant. The BCOP assay reports results for both opacity and permeability of prepared corneas for the test item, a negative control and a positive control (3 replicated per treatment type). Using the In vitro Irritancy Score the test item achieved a value of 142.5, the positive control achieved a value of 116.7 and the negative control achieved a value of 0.5, all of which were within the acceptance criteria of the standard. In conclusion, the test item was assigned a classification of Category 1. UN GHS or EU CLP Causes serious eye damage.