Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 - 27 October 2017

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 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)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

EC Number:

275-085-2

EC Name:

Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

Cas Number:

70983-43-6

Molecular formula:

see structural formula below, for n=4 C13H24N2O3 HO.Na

IUPAC Name:

Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

Test material form:

liquid

Remarks:

oily liquid

Details on test material:

Batch No.: 54542L16
Storage at room temperature, protected from light.

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: ABP, Perth, PH1 3XB
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Cow eyes were collected from freshly slaughtered cattle, and placed into containers containing Hank’s balanced salt solution (HBSS). Transport to test facility was on the same day as slaughter. Cold packs were used to keep the contents cool.
- Time interval prior to initiating testing: Corneas were prepared and used for testing on the day of collection
- indication of any existing defects or lesions in ocular tissue samples: Upon receipt, eyes were rinsed with HBSS and examined for defects (scratches, opacity or neovascularisation) prior to use.Any eyes showing defects were rejected from further use.
- Indication of any antibiotics used: Not specified

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration Test Item: 100 % and 10% v/v in phyisiological saline
NEGATIVE CONTROL: Physiological saline
- Amount(s) applied: 750 µL
- Concentration: Sodium Chloride 0.9%, w/v
POSITIVE CONTROL : Ethanol
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration: 100 % w/v

Duration of treatment / exposure:

10 min ± 1 min

Duration of post- treatment incubation (in vitro):

2h ± 5 min

Number of animals or in vitro replicates:

Three Corneas per test item and 4 corneas per control

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
Upon receipt, eyes were rinsed with HBSS and examined for defects (scratches, opacity or neovascularisation) prior to use. Any eyes showing defects were rejected from further use. Corneas from undamaged eyes were dissected, leaving a sclera margin of ca 3 mm. Collected corneas were placed epithelial side down in HBSS at ambient temperature until use. Prior to mounting corneas the width of the cornea was measured using a ruler.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas were then mounted, epithelial side forward, into pre-warmed corneal holders. Once mounted, both chambers of the cornea holders were filled with pre-warmed EMEM (Eagles Minimum Essential Medium) without phenol red. The posterior chamber was filled first to encourage the cornea to return to its original curvature, and care was taken to avoid the introduction of bubbles into the medium. Holders were then allowed to equilibrate for >1 h in an incubator.
Unless otherwise stated, all incubations of corneas were conducted in an incubator set to maintain 32°C (actual temperatures have been recorded in the raw data). At the end of the equilibration period, the medium in both chambers was replaced with fresh, EMEM without phenol red. Baseline opacity readings were then taken using an Opacitometer (Duratec Analysentechnik GmbH) comprising a Testo 545 luminometer with a photo diode and xenon halogen light source in a custom enclosure. Damaged corneas (opacity >7 opacity units) were rejected from further use.

TREATMENT METHOD: closed chamber. Prior to dosing, cornea holders were tipped forward to avoid contact between the dosing solution and the corneal epithelium. Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts (100% and 10% v/v in physiological saline) (750 µL) was applied to the surface of three corneas using a syringe. The negative control, physiological saline (750 µL), and the positive control, ethanol (750 µL), were also applied to the surface of three corneas each, undiluted, using a syringe. The anterior chambers were then sealed with tape. To begin exposure, the corneal holders were tilted to a horizontal position, taking care to ensure that the entire epithelial surface was covered with the dosing solution. The dosed units were then returned to the incubator for 10 min ± 1 min.

REMOVAL OF TEST SUBSTANCE
EMEM with and without phenol red were pre-warmed prior to use. Following the exposure incubation, the test items and control solutions were removed from the anterior chambers through the holes using a vacuum pump. Imidazolium compounds, 2-C4-8-alkyl-1-(2- carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts (undiluted) could not be removed directly using the vacuum pump, therefore EMEM with phenol red was added to the anterior chamber. This diluted the test item and it was then removed with the vacuum pump.Corneas were then rinsed three times with EMEM with phenol red (ca 5 mL per rinse), removing the EMEM each time, then rinsed once with EMEM without phenol red (ca 5 mL), which was also removed. Finally, both chambers were refilled with EMEM without phenol red, and any bubbles introduced into the medium were removed.

POST-INCUBATION PERIOD: 2 h ± 5 min.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity readings were then taken using an Opacitometer (Duratec Analysentechnik GmbH) comprising a Testo 545 luminometer with a photo diode and xenon halogen light source in a custom enclosure. Gross damage or other changes were observed by visual assessment.

- Corneal pemeability:
Following the measurement of opacity, the EMEM in both chambers of the cornea holder was removed. Posterior chambers were refilled with EMEM without phenol red and sealed. Sodium fluorescein solution (ca 1 mL, 4 mg/mL in EMEM without phenol red) was added to the anterior chambers. Cornea holders were rotated to the horizontal position, and incubated for 90 min ± 5 mins in an incubator set to maintain a temperature of 32°C (actual temperatures have been recorded in the raw data).

At the end of the permeability test, EMEM from the posterior chambers was collected. These samples were stored overnight in a refrigerator set to maintain a temperature of 4°C.
Following overnight storage, triplicate aliquots from each sample (380 µL) were transferred into a 96 well plate, and analysed with a Multiskan Go plate reader at 490 nm. Samples with an OD > 1.5 were diluted and absorbances adjusted accordingly. Three blank wells containing EMEM without phenol red (380 µL) were analysed on the same plate, along with two aliquots of a quality control solution (sodium fluorescein, 5 µg/mL in EMEM without phenol red). Using the standard curve prepared previously these two quality control samples gave acceptable results, of 5.3 µg/mL, when the plate was read.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = Opacity change + (15 x A490 Value)

DECISION CRITERIA:
In accordance with OECD Test Guideline No. 437, irritancy was assigned on the basis of in vitro irritancy scores (IVIS), calculated from the opacity and permeability value for each cornea, as follows:

IVIS<=3: not classified according to GHS/CLP
3IVIS>55: Classified as Eye Damaging Category 1 according to GHS/CLP

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Concentration: 100%

Value:

151.71

Vehicle controls validity:

valid

Remarks:

IVIS: 0.0

Positive controls validity:

valid

Remarks:

IVIS: 87.38

Remarks on result:

positive indication of irritation

Remarks:

UN GHS/CLP Category 1

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Concentration 10% v/v in phisiological saline

Value:

124.83

Vehicle controls validity:

valid

Remarks:

IVIS: 0.0

Positive controls validity:

valid

Remarks:

IVIS: 87.38

Remarks on result:

positive indication of irritation

Remarks:

UN GHS/CLP Category 1

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean post treatment opacity of 3.065 (historical values range 3.56 +/- 1.82) / mean permeability corrected for blank of 0.01 (historical values range 0.016 +/- 0.016). The classification achieved was “No Category”.
- Acceptance criteria met for positive control: yes, mean IVIS 87.38 (historical range: Mean +/- 2SD =42.28 to 98.49). The classification achieved was “Category 1".
- Test item treatement: The BCOP results were similar for all corneas and are presented in full in Table 1 (see Any other information on results incl. tables section).

Any other information on results incl. tables

Table 1                         BCOP Results for Controls and Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-

dihydro-3-hydroxyethyl), hydroxides, sodium salts undiluted and 10% v/v (n=3 and n=4 controls)

Treatment	Opacity Post Dose (Opacity Units, Uncorrected)	Corrected* Opacity Change (Opacity Units)	Permeability (A490), Corrected for Blank and Dilution Only	Permeability (A490), Corrected*	IVIS	IVIS (Mean)	UN GHS Category**
Physiological Saline (Negative Control)	2.25 3.07 3.23 3.46	0.06 -0.17 0.59 -0.49	0.004 0.007 0.005 0.011	-0.003 0.000 -0.001 0.004	0.02 -0.16 0.58 -0.43	0.00	No Category
Ethanol (Positive Control)	32.17 31.11 39.60 35.58	29.98 29.42 36.74 32.42	4.591 2.947 3.094 4.126	4.584 2.940 3.087 4.119	98.74 73.52 83.05 94.20	87.38	Category 1
Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5- dihydro-3-hydroxyethyl), hydroxides, sodium salts (undiluted)	73.62 66.05 102.05	71.14 63.49 100.86	5.215 3.749 5.698	5.208 3.742 5.691	149.26 119.63 186.23	151.71	Category 1
Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5- dihydro-3-hydroxyethyl), hydroxides, sodium salts (10% v/v)	46.18 45.87 55.85	43.32 43.76 52.06	3.979 6.603 5.129	3.972 6.596 5.122	102.90 142.69 128.89	124.83	Category 1

* Corrected for Negative Control

** Classifications from OECD Guidelines for the Testing of Chemicals No. 437 (2017)

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In conclusion, both undiluted and diluted (10% (v/v) in physiological saline) Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts (CAS No. 70983-43-6) was classified as ‘Category 1’ using the BCOP assay according to the UN GHS classifications and the CLP classification system.

Executive summary:

The objective of this study was to evaluate the ocular irritation potential of Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts (CAS No. 70983-43-6) at two concentrations (undiluted and diluted 10% (v/v) in physiological saline) using the Bovine Corneal Opacity and Permeability (BCOP) assay.

Corneas were dissected from freshly obtained cow eyes, the cornea diameter was measured and they were mounted into corneal holders. Following a pre-dose equilibration period, and measurement of baseline opacity (using an opacitometer), the epithelial sides of the corneas

were treated as follows: Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts (undiluted and diluted 10% (v/v) in physiological saline) was applied to three corneas each (ca 750 µL per cornea). Physiological saline or ethanol as negative and positive controls, respectively, were applied to four corneas each (ca 750 µL per cornea). Following exposure for 10 min ± 1 min, the test or control items were rinsed off and corneas underwent a recovery period of 2 h ± 5 min.

The opacity of each cornea was then determined, followed by measurement of permeability by assessment of the passage of sodium fluorescein through the cornea. Irritancy was assigned on the basis of in vitro irritancy scores (IVIS).

The results are summarised as follows:

The mean IVIS score for Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5 - dihydro-3-hydroxyethyl), hydroxides, sodium salts Undiluted and 10% v/v were 151.71 and 124.83 respectively

In conclusion, both undiluted and diluted (10% (v/v) in physiological saline) Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts (CAS No. 70983-43-6) were classified as ‘Category 1’ using the BCOP assay according to the UN GHS classifications and the CLP classification system.