Dibenzoyl-L-tartaric acid monohydrate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1994

Report date:

1993

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dibenzoyl-L-tartaric acid monohydrate

EC Number:

220-374-0

EC Name:

Dibenzoyl-L-tartaric acid monohydrate

Cas Number:

2743-38-6

Molecular formula:

C18H14O8

IUPAC Name:

(2R,3R)-2,3-bis(benzoyloxy)butanedioic acid

Test material form:

solid: particulate/powder

Details on test material:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich, product no. 34,584-9
- Purity: 98%

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich.

OTHER SPECIFICS: To ensure blind experimental conditions, in vitro as well as in vivo, chemicals were sampled, coded, and shipped by an independent Company (MCS-Pharma, Erstein, France).

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes were collected from a commercial abattoir.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): eyes were collected in a plastic jar containing 1 litre HBSS for approximately 25 eyes. Buffer storage and eyes transportation to the laboratories were performed at ambient temperature, and the eyes were used within 2 hr of the killing of the animals.
- Time interval prior to initiating testing: within 2h after collection.
- indication of any existing defects or lesions in ocular tissue samples: no.
- Indication of any antibiotics used: no.

Test system

Vehicle:

other: Minimum essential medium (MEM)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 ml was applied on the epithelium with an appropriate syringe and needle.
- Concentration: Solids were prepared as an approximate 20% solution or suspension (200mg + 1ml) in MEM using a mortar and pestle for homogeneous preparations.

VEHICLE
- Concentration (if solution): Minimum essential medium (MEM) was prepared from powder supplemented with glutamine and sodium bicarbonate as indicated by the supplier, the pH was adjusted to 7.4 and the medium was freshly used or stored refrigerated (1-wk stock).
- Lot/batch no.: Sigma M-3024

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 4 h at 32ºC.

Number of animals or in vitro replicates:

The number of corneas per experiment was 15, including a control group of 3 corneas treated with MEM, and two groups of 6 corneas treated with test compound.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
During dissection great care was taken to avoid damage of corneal surfaces (epithelial and endothelial). Each cornea was carefully examined, and those presenting defects, such as neovascularization, pigmentation, opacity or scratches were discarded. Globes were first dissected free of surrounding tissues and placed in a jar containing fresh HBSS. Selected corneas were dissected with a 2-3-mm rim of sclera for easier handling, and stored in a petri dish containing HBSS until use. Corneas were then mounted in holders filled with MEM, and incubated for 1 hr in a water-bath at 32ºC.

NUMBER OF REPLICATES : 6

NEGATIVE/SOLVENT CONTROL USED : MEM

POSITIVE CONTROL USED : 750 μL of 20% (w/v) imidazole solution in MEM

APPLICATION DOSE AND EXPOSURE TIME : 750 μL of 20% (w/v) test item solution in MEM, 4h exposure.

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times (until the medium was clear) with approximately 4 ml MEM.
- POST-EXPOSURE INCUBATION: 2h at 32ºC.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer (Electro-Design, Riom, France) previously calibrated with standardized opaque sheets of polyester.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (490 nm)
- Others: pertinent visual observations.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = opacity value + 15 times O.D. value

DECISION CRITERIA: score 0 - 25, mild irritant; 25.1 - 55, moderate irritant; ≥ 55.1, severe irritant..

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of results from 11 laboratories, mean of 6 corneas each

Value:

120.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not specified.

DEMONSTRATION OF TECHNICAL PROFICIENCY: not applicable (100% agreement within all laboratories).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Table 1. In vitro scores from individual laboratories for BCOP assay (n=6)

	Score in laboratory no.:													Mean score
Compound	Physical form	1	2	3	4	5	6	7	8	9	10	11	12
Test item 2743-38-6	S	104	134	82	118	110	66	88	193	82	-	213	135	120.5
Imidazole 288-32-4	S	75	73	140	81	96	62	82	122	64	81	114	65	87.9

 

Table 2. Agreement between laboratories.

Compound	Physical form	Mean IVIS	Agreement(%)	Result
Test item 2743-38-6	Solid	120.5	100	Severe irritant
Imidazole 288-32-4	Solid	87.9	100	Severe irritant

Applicant's summary and conclusion

Interpretation of results:

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

Remarks:

EU criteria.

Conclusions:

The test item is severely irritant to the eye (IVIS = 120.5)

Executive summary:

An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted by 12 laboratories in an interlaboratory validation study, in order to determine the potential severe eye damaging effects of the test item. The study was performed according to the OECD guideline 437 under GLP conditions. Sets consisting of 6 corneas each were exposed to either 750 μL of 20% (w/v) of test item suspension in MEM or 750 μL of 20% (w/v) imidazole in MEM (positive control) for 4h at 32ºC; a negative control was carried out in parallel with 3 corneas, treated with MEM. After exposure, opacity and permeability of the corneas was measured, and an in vitro irritancy score was calculated combining both parameters. As a protocol variation, four of the laboratories performed the study with preserved corneas. The mean value from 11 laboratories and the % agreement were calculated and compared to in vivo data. The mean IVIS score values obtained were 87.9 and 120.5 for imidazole and the test item, respectively. Therefore, the test item causes damage to the eye (Cat.1).