Dilithium salicylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2017 - 17 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

26 July 2013

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dilithium salicylate

EC Number:

254-228-2

EC Name:

Dilithium salicylate

Cas Number:

38970-76-2

Molecular formula:

C7H4Li2O3

IUPAC Name:

dilithium salicylate

Test material form:

solid: particulate/powder

Details on test material:

Lot # 17-24168
Expiration: 20 Mar 2019
White Powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ExxonMobil Research & Engineering (Paulsboro, NJ) Lot # 17-24168
- Expiration date of the lot/batch: 20 March 2019
- Purity test date: 04 August 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: test substance was soluble in vehicle throughout duration of study

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: used as received
- Final dilution of a dissolved solid, stock liquid or gel: 200 mg/mL of dilithium salicylate in 0.9% sodium chloride

Test animals / tissue source

Species:

other: Isolated Bovine Cornea

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

The bovine eyes, in a Hanks’ Balance Salt Solution (HBSS) with penicillin-streptomycin, were received from Spear Products on 17 Aug 2017 and transported to MB Research in a refrigerated container.

Test system

Vehicle:

physiological saline

Remarks:

0.9% Sodium Chloride Irrigation

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 ml of the 20% (w/v) test article formulation in saline

Duration of treatment / exposure:

Four hours (±10 minutes) at 32 (±1)°C

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

The solution was removed from the anterior chamber and replaced with 1.0 ml of 0.5% sodium fluorescein solution in Dulbecco's phosphate buffered saline (PBS). Each holder was then returned to the 32 (±1)°C incubator for 90 (±5) minutes.

Number of animals or in vitro replicates:

Three bovine corneas per group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of
vascularization, pigmentation, opacity, or scratches was discarded. Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS.

QUALITY CHECK OF ISOLATED CORNEAS: A pre-exposure determination of opacity was made for each cornea by measuring each against the blank
supplied by the opacitometer. Any cornea with a value greater than 7 units was discarded.

NUMBER OF REPLICATES: 3 isolated bovine corneas per group

NEGATIVE CONTROL USED: Modified Eagles Medium (MEM)

SOLVENT CONTROL USED (if applicable): 0.9% Saline

POSITIVE CONTROL USED: 20% (w/v) imidazole formulation in 0.9% saline

APPLICATION DOSE AND EXPOSURE TIME: A volume of 0.75 ml of the 20% (w/v) test article formulation in 0.9% saline for four hours (±10 minutes). All holders and corneas were placed in a horizontal position (anterior side up) in the 32 (±1)°C incubator

TREATMENT METHOD: All corneas were dosed via the closed-chamber method.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Test article formulation, 20% imidazole formulation, MEM or 0.9% saline was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Differences in light transmission through the control and treated cornea were determined using an OP-KIT opacitometer produced by Electro-Design Corporation of Riom, France. Each treated cornea will be scored in comparison to the blanks provided with the OP-KIT opacitometer .
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD450) Milton Roy/ Spectronic 20-D Colorimeter Model: 333175 Serial No: 33241700
- Others (e.g, pertinent visual observations, histopathology): none

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS Less than or equal to 3 = GHS No Category
IVIS Greater than 3 and less than or equal to 55 = No prediction can be made
IVIS Greater than 55 = Substance causing serious eye damage (GHS Category 1)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

4 hour

Value:

110.34

Vehicle controls validity:

valid

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:

In vitro irritation score (IVIS) calculated from opacity score

Value:

128.75

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Corneal surface sloughing was reported in one of three cornea after 4 hour exposure.

The Imidazole positive control IVIS was 91.02, which fell within the acceptance range of 58.10 – 135.86 (± 2 standard deviations of the historical mean).

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The mean corneal opacity, and in-vitro irritation score for Dilithium Salicylate were 110.34 and 128.75, respectively. These findings warrant classification of Dilithium Salicylate as a substance inducing serious eye damage (Category 1 Eye Irritant) under the Regulation (EC) 172/2008 on classification, labelling and packing of substances and mixtures (CLP).

Executive summary:

Dilithium Salicylate was applied to isolated bovine cornea to assess for the potential to induce serious eye damage and irritation. Ocular damage was assessed at 4 hours post-instillation and scored through measurement of corneal opacity and permeability. The mean corneal opacity, and in-vitro irritation score for Dilithium Salicylate were 110.34 and 128.75, respectively. These findings warrant classification of Dilithium Salicylate as a substance inducing serious eye damage (Category 1 Eye Irritant) under the Regulation (EC) 172/2008 on classification, labelling and packing of substances and mixtures (CLP).