Calcium dibenzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-08 to 2018-01-30

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 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2017-10-09

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Test material

Test material information

Constituent 1

Reference substance name:

Calcium dibenzoate

EC Number:

218-235-4

EC Name:

Calcium dibenzoate

Cas Number:

2090-05-3

Molecular formula:

C7H6O2.1/2Ca

IUPAC Name:

calcium dibenzoate

Test material form:

solid: particulate/powder

Details on test material:

- State of aggregation: solid powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry; < 30 °C

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Age at study initiation: animal #1: approx. 31 weeks old; animal #2: approx. 40 weeks old
- Weight at study initiation: animal #1: 4.2 kg; animal #2: 4.3 kg
- Housing: individually housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm²
- Diet (ad libitum): autoclaved hay and Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3 °C
- Relative humidity: 55 ± 10%
- Air changes: at least 10x / hour
- Photoperiod (hrs dark / hrs light): 12/12

Test system

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g of the test item in the conjunctival sac of one eye. Untreated eye served as control.

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

animal #1: 1, 24, 48 and 72 hours as well as 4, 5, 6, 7, 8 and 9 days after test item application
animal #2: 1, 24, 48 and 72 hours as well as 4, 5, 6 and 7 days after test item application

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

2 male rabbits

Details on study design:

PREPARATION OF THE ANIMALS
Within 24 hours before the test and immediately prior to the application both eyes of each animal were examined.
Approx. 23 hours before the application the eyes were examined with the aid of a fluorescein solution (Fluoreszein SE Thilo®). The eyes were rinsed with physiological saline 0.9 % NaCl after the examination. None of the animals showed eye irritation, ocular defects, or pre-existing corneal injury.

INITAL TEST AND CONFIRMATORY TEST
The in vivo test was performed initially using one animal. The results of the initial test did not indicate the test item to be corrosive or a severe irritant to the eye using the procedure described. In order to confirm the response, one additional animal was treated in the same manner.

USE OF TOPICAL ANESTHETICS AND SYSTEMIC ANALGESICS
One hour before the application of the test item, 0.01 mg/kg of buprenorphine (Temgesic® 0.3 mg/mL) was administered subcutaneously in order to achieve a therapeutic level of systemic analgesia. Approx. 5 minutes prior to the application of the test item, 1-2 drops of an ocular anaesthetic (Proparakaine-POS® hydrochloride ophthalmic 0.5% solution) were administered in both the treated and the control eye of each animal.
To prevent pain and distress after the application of the test item both animals were treated with doses of buprenorphine and meloxicam (Metacam® 5 mg/mL) to provide a continued therapeutic level of systemic analgesia. Treatment with the analgesic medication was conducted from 11 hours post-application (day 0) upto 4 or 5 days post-application.

REMOVAL OF TEST SUBSTANCE
- Washing: treated eyes were rinsed with physiological saline 0.9 % NaCl.
- Time after start of exposure: 1 hour after the application

SCORING SYSTEM: according to Draize scale

TOOL USED TO ASSESS SCORE: slit lamp biomicroscope / fluorescein
24 hours post-application and from then on daily until end of the observation period, the treated eyes were was examined with the aid of a fluorescein solution and a slit lamp biomicroscope. After the examination the eyes were was rinsed with physiological saline 0.9 % NaCl.

OBSERVATIONS
- body weight: prior to the administration and at least at the end of the observation period
- clinical observations: nature, severity and duration were recorded

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Results 2

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible within: 48 hours

Results 3

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

2.33

Max. score:

3

Reversibility:

fully reversible within: 9 days

Results 4

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 5 days

Results 5

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Results 6

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Results 7

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Results 8

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 4 days

Irritant / corrosive response data:

Animal #1:
- conjunctival redness: one hour post-application slight conjunctival redness (grade 1) was observed. 24 hours after application severe conjunctival redness (grade 3) was observed. From 48 hours until 72 hours post-application a moderate conjunctival redness (grade 2) was observed, which changed to a slight conjunctival redness (grade 1) from day 4 until day 8 post-application. The effect was fully reversible within day 9 after application of the test material.

- conjunctival chemosis: one hour and 24 hours after application a moderate conjunctival chemosis (grade 2) was observed. Between 72 hours and day 4 after application the animal showed a slight conjunctival chemosis (grade 1). The effect was fully reversible within day 5 after application of the test material.

- iris: 24 hours post-application the animal showed a slight iris lesion (grade 1). The effect was fully reversible within 48 hours after application of the test material.

- cornea opacity: one hour until 48 hours post-application slight corneal opacity (grade 1) was found in the animal. The effect was fully reversible within 72 hours after application of the test material.

Animal #2:
- conjunctival redness: moderate conjunctival redness (grade 2) was observed one hour after application as well as on day 4 and day 6 post-application. A severe conjunctival redness (grade 3) was observed in the animal from 24 hours until 72 hours post-application and on day 5 post-application. The effect was fully reversible within day 7 after application of the test material.

- conjunctival chemosis: a moderate conjunctival chemosis (grade 3) was detected in the animal one hour post-application before severity level decreased 24 hours after application (grade 2) and from 48 until 72 hours post-application (slight conjunctival chemosis; grade 1). The effect was fully reversible within day 4 after application of the test material.

- iris: slight iris lesion (grade 1) was observed in the animal from one hour until 48 hours after application. The effect was fully reversible within 72 hours after application of the test material.

- cornea opacity: slight corneal opacitiy (grade 1) was found in the animal from one hour until day 6 post-application. The effect was fully reversible within day 7 after application of the test material.


Local effects were observed in both animals. The observed local effects mainly comprise slight to severe hypersecretion, a dark red nictitating membrane, and a dark red spot on the nictitating membrane. All these findings were reversible within 5 or 6 days post-application.
Upon fluorescein examinations starting 24 hours post-application, the treated eyes of both animals showed corneal lesions (starting with approx. 75% of the area) which were completely reversible within 7 days.

Other effects:

- clinical observations: neither mortalities nor significant clinical signs of toxicity were observed.
- body weight: the body weight development of one rabbits was within the expected range, while the other animal showed slight weight loss.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The substance is irritating to the eyes.
According to the EC Regulation No. 1272/2008 and subsequent amendments, Calcium dibenzoate is classified as an eye irritant (Category 2; H319).