1-phosphonobutane-1,2,3,4-tetracarboxylate and 2-phosphonosuccinate sodium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 20/11/2015 to 08/02/2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant study performed according to an internationally-recognized guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Remarks:

2015-06-04

Test material

Test material information

Constituent 1

Details on test material:

Name: 1-phosphonobutane-1,2,3,4-tetracarboxylate and 2-phosphonosuccinate sodium salts

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

Chicken head collection and transport:
Strain of chicken: ROSS 308
Source: TARAVIS Ltd. (Address: H-9600 Sárvár, Rábasömjéni út. 129., Hungary)
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old and 2.1 kg) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with physiological saline, then placed in a in a lockable plastic box (4-5 heads per box). The heads were transported to CiToxLAB Hungary Ltd. and processed within approximately 2 hours after collection.


Eyes selection
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Eyes examination and acclimatization time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head) while avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

The appropriate number of eyes was selected and placed in the superfusion apparatus. After that, the eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to clearly see the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

Identification
The eyes were identified by chamber number, marked on the door of the chamber

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: physiological saline 0.9%

Amount / concentration applied:

After the zero reference measurements, the eye (in its retainer) was removed from the chamber, held in a horizontal position and the test item was applied onto the centre of the cornea, taking care not to damage or touch the cornea.

A volume of 30 µL of test item was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance.

Duration of treatment / exposure:

The time of application: 10 secondes

Observation period (in vivo):

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit BP 900 slit-lamp microscope was used for the measurements

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item (or controls), if possible. The eye was returned to the chamber after rinsing. The time while the eye was out of the chamber was limited to the minimum.

SCORING SYSTEM: The endpoints evaluated are corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium).
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 substance

Positive Control
Name: Benzalkonium chloride solution, 50% in water
Batch number: SZBC2960V
CAS Number: 63449-41-2
Expiry date: 25 March 2018
Manufacturer: Sigma-Aldrich Co.
Storage condition: Room temperature
This material was diluted with distilled water (Supplier: Teva Co., Lot number: 6820914, Expiry date: 30 September 2017) to achieve the final concentration of 5% (w/v). The treatment solution was prepared immediately before the experiment.

Negative Control
Name: Physiological saline (0.9% (w/v) NaCl solution)
Batch number: 51642Y05-1
Manufacturer: B. Braun Pharmaceuticals SA
Expiry date: 31 March 2018
Storage conditions: Room temperature
Quality: Sterile

Fluorescein retention test
Name: Fluorescein, 10% (w/v) solution
Batch number: 13221042
Manufacturer: Delpharm Huningue SAS
Expiry date: 30 April 2016
Storage conditions: Room temperature
This material was mixed with physiological saline (Supplier: B. Braun Pharmaceuticals SA, Lot No: 51642Y05-1, Expiry Date: 31 March 2018) to achieve the final concentration of 2% (w/v). The resulted solution was stored at room temperature (Dispensary code: S43090, Expiry date: 02 January 2016).

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: Corneal swelling

Basis:

mean

Time point:

other: 75 min

Score:

0

Results 2

Irritation parameter:

other: Corneal Swelling

Basis:

mean

Time point:

other: 240 min

Score:

ca. 0

Results 3

Irritation parameter:

cornea opacity score

Remarks:

Opacity

Basis:

mean

Score:

0.17

Results 4

Irritation parameter:

other: Fluorescein retention

Basis:

mean

Score:

0

Any other information on results incl. tables

Test item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	-0.5%	I
Mean maximum corneal opacity	0.17	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3xI

Based on thesein vitroeye irritation assays in isolated chicken eyes with 1-phosphonobutane-1,2,3,4-tetracarboxylate and 2-phosphonosuccinate sodium salts (EC number: 942-790-8), the test item was not irritant (No Category), UN GHS Classification: Not classified

Positive Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	10.5%	II
Mean maximum corneal swelling at up to 240 min	25.7%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Loosening of epithelium was observed in two eyes at 75 minutes and in one eye at 180 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

The positive control (5% (w/v) Benzalkonium chloride solution)was classified as severely irritating,UNGHS Classification: Category 1.

Negative Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3xI

The negative control (Physiological saline)was classified as non-irritating, UN GHS Classification:Non-classified.

Validity of the test

The results from all eyes used met the quality control standards. The negative control and positive control results were within historical control data. The experiment was considered to be valid.

 

Historical Control data (updated on 24 September 2015):

 

Negative Control: Physiological Saline

 

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-3.2 %	3.4 %
Maximum corneal swelling at up to 240 min	-4.8 %	3.4 %
Maximum corneal opacity change	0.00	0.50
Fluorescein retention	0.00	0.50
Number of studies	200

 

Positive Control: 5% (w/v)Benzalkonium chloride soultion

 

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-8.5 %	27.0 %
Maximum corneal swelling at up to 240 min	-10.7 %	34.8 %
Maximum corneal opacity change	2.50	4.00
Fluorescein retention	1.50	3.00
Number of studies	108

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

Based on these in vitro eye irritation assays in isolated chicken eyes with 1-phosphonobutane-1,2,3,4-tetracarboxylate and 2-phosphonosuccinate sodium salts (EC number: 942-790-8), the test item was not irritant (No Category), UN GHS Classification: Not classified

Executive summary:

In vitro eye irritation test in isolated Chichen Eyes with 1-phosphonobutane-1,2,3,4-tetracarboxylate and 2-phosphonosuccinate sodium salts was realised according to the OECD 438 guideline and under GLP conditions.

1-phosphonobutane-1,2,3,4-tetracarboxylate and 2-phosphonosuccinate sodium salts was a colourless to yellow aqueous solution with a purity of 97.1%. 30µL of the substance was applied on top isolated eyes for an exposure period of 10 seconds and the surface of the cornea was rinsed thoroughly with 20 mL of physiological saline at ambiant temperature.

No signs of irritation with the test item were describe during the test.

Negative and positive controls were within the historical data.

 

The test item was not classified according to UN GHS classification.