β,β-carotene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 20 February 2018 Experimental completion date: 02 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD guideline and performed under GLP

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 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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

β,β-carotene

EC Number:

230-636-6

EC Name:

β,β-carotene

Cas Number:

7235-40-7

Molecular formula:

C40H56

IUPAC Name:

β,β-carotene

Details on test material:

Identification: beta-carotene
Batch: WC01607211
Purity: 100.4% (UV, dried)
Appearance: Brown-red, crystalline powder
Expiry Date: 19 July 2018
Storage Conditions: In the refrigerator, light protected, under N2-atmosphere

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Freshly isolated bovine cornea (at least 9 month old donor cattle)
Rationale: OECD 437
Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany

Collection of Bovine Eyes
Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% suspension (w/v) in saline using sonication for 10 minutes.

VEHICLE (Saline)
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9% NaCl in deionised water


POSITIVE CONTROL (Benzalkonium chloride)
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 10% (w/v) in 0.9% (w/v) NaCl (saline) using sonication for 10 minutes.

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3 for test itema and each control

Details on study design:

Preparation of Corneae
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and for the negative and positive controls, respectively.
 
Exposure of the Corneae to the Test Groups
The anterior compartment received the test item suspension or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneae, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted 240 minutes.
Afterwards, the test item or the control items, respectively, were each rinsed off from the according application sides with saline, and fresh incubation medium was added into the anterior compartment and opacity was measured (t240).
In the second step of the assay, permeability of the cornea was determined.

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the corneae to the different test groups, and after rinsing the opacity value was determined again (t240).

Permeability Determination
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

2.74

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

This in vitro study was performed to assess the corneal damage potential of beta-carotene by means of the BCOP assay using fresh bovine corneae.
After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline of the test item beta-carotene, the positive, and the negative controls were applied to the different corneae and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneae and opacity was measured again (t240).
After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.
The experiment has to be repeated twice since the values of the controls did not fulfil the acceptance criteria.
For the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.03).
The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =108.55) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
Relative to the negative control, the test item beta-carotene did not cause a relevant increase of the corneal opacity. The calculated mean IVIS was 2.74 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not categorized.

Any other information on results incl. tables

Results after 240 Minutes Treatment Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score
		Mean		Mean
Negative Control	1	0.33	0.045	0.046	1.68	1.03	No Category
	0		0.044		0.66
	0		0.050		0.75
Positive Control	95.67*		0.627*		105.07	108.55	Category 1
	103.67*		0.735*		114.69
	98.67*		0.483*		105.91
beta-carotene	2.67*		0.030*		3.11	2.74	No Category
	1.67*		0.035*		2.19
	2.67*		0.017*		2.92

*corrected values

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, beta-carotene is not classified for eye irritation (GHS).

Executive summary:

This in vitro study was performed to assess the corneal damage potential of beta-carotene by means of the BCOP assay using fresh bovine corneae. Testing was performed under GLP and in compliance with the test guidelines OECD 437 and EC B.47.

After a first opacity measurement of the fresh bovine corneae (t₀), the 20% (w/v) suspensionin saline (0.9% (w/v) NaCl in deionised water) of the test item beta-carotene as well as the positive and the negative controls were each applied to different corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae andopacity was measured again (t₂₄₀).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (CLP/EPA/GHS (Cat 1)).

 

Relative to the negative control, the test item beta-carotene did not cause a relevant increase of the corneal opacity. The calculated mean in vitro irritancy score was 2.74. According to OECD 437 the test item is not classified for eye irritation (GHS).