2,2'-(piperazine-1,4-diyl)bis(ethanesulphonic) acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-06-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July 2013

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)

Version / remarks:

08 December 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(piperazine-1,4-diyl)bis(ethanesulphonic) acid

EC Number:

227-057-6

EC Name:

2,2'-(piperazine-1,4-diyl)bis(ethanesulphonic) acid

Cas Number:

5625-37-6

Molecular formula:

C8H18N2O6S2

IUPAC Name:

2,2'-piperazine-1,4-diyldiethanesulfonic acid

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary (slaughter house)
- Number of animals: not specified
- Characteristics of donor animals: not specified
- Storage, temperature and transport conditions of ocular tissue: After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box). The ambient temperature was optimal (19.4 ºC to 20.2 ºC) during the transport.
- Time interval prior to initiating testing: Heads were transported for use approximately within 2 hours from collection.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes were evaluated before removal from heads (using fluorescein solution) and afterwards (see below). Only eyes in good condition were used for the experiment.
- Indication of any antibiotics used: not specified
- Selection and preparation of corneas: Please refer to details on study design.
- Quality check of the isolated corneas: Please refer to details on study design.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.03 g (per eye)

CONTROLS
- Amount applied: 0.03 g imidazole; 30 µL NaCl solution

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

4 hours

Number of animals or in vitro replicates:

3 eyes (test substance and positive control)
1 eye (negative control)

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyeball was carefully removed from the orbit. The procedure avoided pressure on the eye 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. 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). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied 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 saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 5 drops/minutes. 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 after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic 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 using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm. 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.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Changes in thickness were not observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

OBSERVATION PERIOD
The control 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 ±5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with approximately 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The cornea opacity was measured at all time points.
- Damage to epithelium based on fluorescein retention: Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.
- Swelling: The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm.
- Macroscopic morphological damage to the surface: Damages eyes were discarded before experiment. Eyes were checked for damage due to the experiment.
- Others (e.g, histopathology): No histopathology evaluation was performed.

SCORING SYSTEM:
- Mean corneal swelling (%): according to TG
- Mean maximum opacity score: according to TG
- Mean fluorescein retention score at 30 minutes post-treatment: according to TG

DECISION CRITERIA: Decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum, up to 240 min

Value:

7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

28

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Mean maximum

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

4.0

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

3.0

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not applicable

Any other information on results incl. tables

Table 1: Results for the test item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	6 %	II
Mean maximum corneal swelling at up to 240 min	7 %	II
Mean maximum corneal opacity	2.0	III
Mean fluoresin retention	0.5	I
Other Observations	None
Overall ICE class	1xI, 1xII, 1xIII

 

Table 2: Results for positive control Imidazole

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	21 %	III
Mean maximum corneal swelling at up to 240 min	28 %	III
Mean maximum corneal opacity	4.0	IV
Mean fluoresin retention	3.0	IV
Other Observations	Cornea opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse.
Overall ICE class	1xIII, 2xIV

The positive control Imidazole was classed as corrosive/severely irritating, UN GHS Classification: Category 1. 

 

Table 3: Results for the negative control NaCl (9 g/L saline)

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0 %	I
Mean maximum corneal swelling at up to 240 min	0 %	I
Mean maximum corneal opacity	0.0	I
Mean fluoresin retention	0.0	I
Other Observations	None
Overall ICE class	3xI

 

Based on the overall ICE Class the negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.

Positive and negative control values were within the corresponding historical control data ranges.

 

Table 4: Historical control data of Positive control (Period of 2011 - 2015)

IMIDAZOLE HISTORICAL CONTROL Dose level: 30 mg / eye

n=168	Relative obobservation time (m(min)	Corneal thickness					Corneal opacity score						Fluorescein retention
		30	75	120	180	240		30	75	120	180	240		∆FR
Maximium	swelling (%):	34	45	49	54	44	Max. OS:	4.0	4.0	4.0	4.0	4.0	Max. FR:	3.0
Minimum swelling (%):		3	9	12	14	15	Min. OS:	2.8	3.3	3.5	3.5	3.5	Min. FR:	2.7
Average:		19	27	31	35	37	Average:	3.7	3.9	3.9	3.9	3.9	Average:	3.0

 

Table 5: Historical control data of Negative control (Period of 2011 - 2015)

NaCl (9 g/L saline) SOLUTION HISTORICAL CONTROL Dose level: 30 µL / eye

n=120	Relative obobservation time (m (min)	Corneal thickness					Corneal opacity score						Fluorescein retention
		30	75	120	180	240		30	75	120	180	240		∆FR
Maximum	swelling (%):	3	3	3	4	3	Max. OS:	0.5	0.5	0.5	0.5	0.5	Max. FR:	0.5
Minimum swelling (%):		0	0	0	0	0	Min. OS:	0	0	0	0	0	Min. FR:	0.0
Average:		0.1	0.3	0.3	0.3	0.3	Average:	0.0	0.0	0.0	0.0	0.0	Average:	0.0

Remark:

n = number of examined eyes

∆FR = Difference between fluorescein retention and fluorescein retention reference value

OS = Opacity score

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xI, 1xII, 1xIII.

Executive summary:

A study according to OECD 438 was conducted with the test item. The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes and rinsed after 10 seconds with saline. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points. The Imidazole (positive control) was ground before use in the study. The test item and positive control were applied in an amount of 30 mg/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with approximately 20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xI, 1xII, 1xIII. Positive and negative controls showed the expected results. The experiment was considered to be valid.