Short Chain Fructo Oligosaccharides

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

short chain Fructo-Oligosaccharides

EC Number:

908-300-1

Molecular formula:

C6H11O5(C6H10O5)nOH

IUPAC Name:

short chain Fructo-Oligosaccharides

Test material form:

solid: particulate/powder

Details on test material:

Identification: Actilight 950P
Substance: Short-chain fructo-oligosaccharides
EC No.: 908-300-1
Appearance: White powder
Purity: 96%

COMPOSITION:
scFOS is short chains of fructose molecules linked to a molecule of sucrose (glucose-fructose disaccharide). Thus, scFOS is a multiconstituent substance composed of three oligosaccharides: 1-kestose (GF2), nystose (GF3) and fructosyl-nystose (GF4).
With:
- TOTAL FOS: 96% expressed as dry matter
- Kestose (GF2): 37.1% FOS
- Nystose (GF3): 50.5% FOS
- 1F-Fructofuranosylnystose (GF4:) 12.4% FOS
- Glucose +Fructose + Saccharose : 4.0% expressed as dry matter

Specific details on test material used for the study:

• Sponsor’s identification: ACTILIGHT® 950P
• Form: powder
• Colour: White
• Storage : room temperature

Test animals / tissue source

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source:
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight):
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples:
- Indication of any antibiotics used:

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg of the test item was applied, as supplied, to the cornea, such that the entire surface of the cornea is evenly covered with the test item.

Duration of treatment / exposure:

The test item was applied for 10 seconds

Number of animals or in vitro replicates:

to 3 enucleated chicken eyes

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Source, collection and transport of chicken eyes:
The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay.
The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).

Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 19 May 2011 at 8:30 am.

Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient tetnperature in polystyrene boxes humidified with toz'els moistened with isotonic saline.
The eyes were enucleated at Phycher on 19 May 2011 at 9:40 am


Preparation of eyes:
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.

The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the isotonic saline drip. The Ghambers of the superfusion apparatus was temperature controlled at 32°C 1.5°C.

After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% fi om the mean value for all eyes are to be rejected.

EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes have been examined and approved, the eyes were incubated between 60 and 76 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time= 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES : to 3 enucleated chicken eyes

APPLICATION DOSE AND EXPOSURE TIME AND REMOVAL OF TEST SUBSTANCE:
Test item:
Immediately following the zero reference measurements, the eye (in its holder) was removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, as supplied, to the cornea, such that the entire surface of the cornea is evenly covered with the test item.

The test item was applied for 10 seconds and then rinsed from the eye with 20 mL of isotonic saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

Controls:
Concurrent negative control (physiological saline — Cooper, Batch No. 19OC31GE) and positive control (sodium hydroxide — Sigma, Batch No. MKBF9973V) were included in this experiment.

METHODS FOR MEASURED ENDPOINTS:
Treated corneas are evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (d 5 minutes) after the post-treatment rinse.
The endpoints evaluated are corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of ftuorescein retention (which is determined only at pretreatment and 30 minutes after test item exposure) are determined at each of the above time points.
The eyes were not kept in an appropriate fixative at the end of the study for possible histopathological examination.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Value:

1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

fluorescein retention score

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

percent corneal swelling

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as corrosive/severe irritant, as expected.

The combination of the three endpoints for the negative control, physiological saline solution, was 2 x I, 1 x II. Therefore, the negative control is classified as non corrosive/severe irritant, as expected.

Any other information on results incl. tables

Results from corneal opacity, swelling, and fluorescein retention are evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint are then combined to generate an Irritancy Classification for each test item.

Once each endpoint has been evaluated, ICE classes can be assigned based on a predetermined range. Interpretation of corneal thickness, opacity, and fluorescein retention using four ICE classes is done according to the scales of the OECD 438 guideline.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, the ocular reactions observed in eyes treated with the test item were slight to moderate:
- maximal mean score of corneal opacity: 1.5, corresponding to the ICE class II;
- mean score of fluorescein retention: 1.2, corresponding to the ICE class II;
-maximal mean corneal swelling: +4%, corresponding to the ICE class I.

Executive summary:

The aim of the study was to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

 

The test item ACTILIGHT 950P was applied, as supplied, at the dose of 30 mg, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes 1'ere rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test substance were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. The experimental protocol was established in accordance with the OECD guideline No 438 adopted 07 September 2009 and the test method B.48 — Commission Regulation (EU) No. 1152/2010 dated 08 December 2010.

The ocular reactions observed in eyes treated with the test item were slight to moderate:

- maximal mean score of corneal opacity: 1.5, corresponding to the ICE class II;

- mean score of fluorescein retention: 1.2, corresponding to the ICE class II;

-maximal mean corneal swelling: +4%, corresponding to the ICE class I.

 

The combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as corrosive/severe irritant, as expected.

 

The combination of the three endpoints for the negative control, physiological saline solution, was 2 x I, 1 x II. Therefore, the negative control is classified as non corrosive/severe irritant, as expected.

 

The results obtained, under these experimental conditions, enable to conclude that the test item ACTILIGHT 950P must not be classified in category 1 “irreversible effects on the eye in accordance with the Regulation (EC) No. 1272/2008.

 

Furthermore, according to the OECD guideline 438 adopted the 25 june 2018 when the combinations of the 3 endpoints is 3 x I, 2 x I, 1 x II or 2 x II, 1 x I, no UN GHS classification is needed. Therefore, test item ACTILIGHT 950P is not requiring classification (UN GHS classification category)