Butanedioic acid, 2-sulfo-, 1(or 4)-[2-(C12-18 and C18-unsatd. amido)ethyl] esters, disodium 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:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to GLP and valid methods and is considered relevant and reliable for classification. It is an in vitro study by means of BCOP, predicting severe eye irritation, which is considered adequate in combination with the in vitro irritation testing.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICCVAM BCOP test method, 2007

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Note for Guidance on Non-Clinical Tolerance Testing of Medicinal Products, CPMP/SWP/2145/00, adopted March 1, 2001.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-18(even numbered) and C18 unsaturated)alkyl)amino]ethyl]esters, disodium salts

IUPAC Name:

Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-18(even numbered) and C18 unsaturated)alkyl)amino]ethyl]esters, disodium salts

Test material form:

other: liquid

Test animals / tissue source

Species:

other: Bovine eyes from cattie in the age range of 6 to 12 months were obtained from Hubert Bahlmann GmbH & Co., Lindern.

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse: Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG 49699 Lindern, Germany
- Characteristics of donor animals (e.g. age, sex, weight): cattle in the age range of 6 to 12 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing 1% Penicillin/Streptomycin . Upon arrival at the laboratory the eyes were examined for defects such as but not limited to increased opacity, scratches and neovascularisation. Only corneas from eyes free of defects were used.
- Time interval prior to initiating testing: > 1 hour
The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM) , while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.
After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer (BASF OP-3.0). A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative (or solvent) control corneas. The remaining corneas were then distributed into treatment and positive control groups.
- Indication of any existing defects or lesions in ocular tissue samples: Only corneas from eyes free of defects were used.
- Indication of any antibiotics used: Hanks’ Balanced Salt Solution (HBSS) containing 1% Penicillin/Streptomycin

Test system

Vehicle:

physiological saline

Remarks:

0.9% NaCl

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration (if solution): The test item was used as a 3.98-fold dilution in 0.9 % NaCl-solution (in order to obtain a 10% w/v dilution of active ingredient, which complies with the description for surfactants).

VEHICLE
- Concentration (if solution): 0.9% NaCl-solution

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours . After rinsing, the corneas were incubated at 32 ± 1 °C for two hours. After this post-exposure incubation period, the corneas were examined.

Number of animals or in vitro replicates:

3 corneas for each treatment group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer (BASF OP-3.0). A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative (or solvent) control corneas. The remaining corneas were then distributed into treatment and positive control groups.

NUMBER OF REPLICATES 3

NEGATIVE CONTROL USED
0.9% NaCl solution
Batch no. 12371453; B. Braun Melsungen AG, 34212 Melsungen, Germany

SOLVENT CONTROL USED (negative control is also solvent control)

POSITIVE CONTROL USED
1% NaOH solution
Batch no. B 3130; Honeywell Specialty Chemicals Seelze GmbH, 30926 Seelze Germany

APPLICATION DOSE AND EXPOSURE TIME
750 µL (of the test or control items)
Exposure period of 10 minutes

TREATMENT METHOD: [closed chamber / open chamber] Not provided.

POST-INCUBATION PERIOD: yes. The corneal holder was equilibrated at 32±1°C for at least one hour.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least 4 times.
After the exposure period of 10 minutes the exposure solution was removed from each chamber and the epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

- POST-EXPOSURE INCUBATION:
After rinsing, the corneas were incubated at 32±1°C for two hours.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry / visible light spectrophotometer using a standard 1 cm path length (OD492)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean permeability OD492 value)

DECISION CRITERIA:
A test item that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.
As stated in the OECD guideline, if the test substance is not identified as an ocular corrosive or severe irritant, additional testing should be conducted for classification and labelling purposes. The BCOP test method has an overall accuracy of 79% (113/143) to 81% (119/147), a false positive rate of 19% (20/103) to 21% (22/103), and a false negative rate of 16% (7/43) to 25% (10/40), when compared to in vivo rabbit eye test method data classified according to the EPA, EU, or GHS classification systems. When substances within certain chemical (i.e., alcohols, ketones) or physical (i.e., solids) classes are excluded from the database, the accuracy of BCOP across the EU, EPA, and GHS classification systems ranges from 87% (72/83) to 92% (78/85), the false positive rates range from 12% (7/58) to 16% (9/56), and the false negative rates range from 0% (0/27) to 12% (3/26).

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

test item

Value:

21.9

Negative controls validity:

valid

Remarks:

mean IVIS 0.03

Positive controls validity:

valid

Remarks:

mean IVIS 87.43

Remarks on result:

other: A test item that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative or solvent/vehicle control responses should result in opacity and permeability values, that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. Upper limit of acceptance for opacity 1.259; Upper limit of acceptance for permeability 0.049.
- Acceptance criteria met for positive control: A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean.
Lower limit of acceptance 76.8; Upper limit of acceptance 133.2.

Any other information on results incl. tables

Table 1. In vitro irritancy score (IVIS)

	Cornea No.	Opacity	Permeability	IVIS
				Per Cornea	Per Group
					Mean	SD
NaCl 0.9% NC	1	-0.066	0.0160	0.2	0.03	0.15
	2	-0.235	0.0130	0.0
	3	-0.262	0.0130	-0.1
NaOH 1%	4	53.497	1.851	81.3	87.43	9.11
	5	65.173	2.181	97.9
	6	51.047	2.137	83.1
Active ingredient 1:10#	7	0.628	1.128	17.5	21.90	3.83
	8	2.295	1.479	24.5
	9	4.854	1.256	23.7

 

NC: negative control

SD: standard deviation

#: tested as 10% concentration in 0.9% NaCl solution

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts, tested in the in vitro BCOP test method, had an IVIS value of < 55.1 and consequently it is not classified as a severe irritant and is not corrosive.

Executive summary:

The purpose of this study was to determine if the test item can be classified as “ocular corrosive and severe irritant” employing an in vitro system. The BCOP test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability in isolated corneas from bovine eyes. The corneas of the eyes were dissected and the remaining sclera was mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers.

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. The measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of the test item.

Three corneas were used for each treatment group (test item, negative and positive controls). The test item was used as a 3.98-fold dilution in 0.9% NaCl-solution in order to obtain a 10% w/v dilution of active ingredient, which complies with the guideline requirements for surfactants. 0.9% NaCl solution was used as the negative control and 1% NaOH solution as the positive control item. The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder for an exposure time of 10 minutes.

The optical density (OD₄₉₂) or absorbance values were measured at a wavelength of 492 nm. An opacity value of 2.592 and a permeability value of 1.288 compared to the negative control were determined. An IVIS 21.90 was calculated. Hence, the test item was not classified as a severe irritant and not corrosive, based on the results of this test.

The corneas treated with the positive control item 1% NaOH solution revealed an opacity value of 56.572 and a permeability value of 2.056 compared to the negative control. The IVIS value of 87.43 was well above the cut-off value of 55.1 and, hence, the acceptance criteria for the test were fulfilled. 1% NaOH solution was a severe irritant and corrosive to eyes.

Under the present test conditions Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts, tested in the in vitro BCOP test method, had an IVIS value of < 55.1 and consequently it is not classified as a severe irritant and is not corrosive.