Amines, C12-16 alkyl dimethyl, reaction products with ethyl chloroacetate, 2-(2-aminoethylamino)ethanol and 2-propenoic 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:

October/November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

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 2

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

26 July 2012 Date of Signature: 11 April 2013

Test material

Test material information

Constituent 1

Reference substance name:

(2-carboxyethyl)({2-[(2-carboxyethyl)(2-hydroxyethyl)amino]ethyl})azanium; [({2-[(2-carboxyethyl)(2-hydroxyethyl)amino]ethyl}carbamoyl)methyl](dodecyl)dimethylazanium; ethanol

IUPAC Name:

(2-carboxyethyl)({2-[(2-carboxyethyl)(2-hydroxyethyl)amino]ethyl})azanium; [({2-[(2-carboxyethyl)(2-hydroxyethyl)amino]ethyl}carbamoyl)methyl](dodecyl)dimethylazanium; ethanol

Test material form:

other: liquid

Test animals / tissue source

Species:

other: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

a volume of 0.75 mL of the undiluted test item was applied on the surface of the bovine corneae.

Duration of treatment / exposure:

The incubation time lasted 10 minutes.

After the test item or control items, respectively, were rinsed off from the application side with saline, the corneae were incubated for further two hours in a vertical position.

Observation period (in vivo):

approx. 130 minutes in total

Number of animals or in vitro replicates:

3 corneae for the test item plus 3 corneae for the negative and positive control, respectively.

Details on study design:

This test is designed to measure the opacity of the cornea by quantifying the ability of light to pass through it. The permeability, as a result of the irritation potential of the test item, was determined using Na-fluorescein solution. The comparison of the opacity before and after the exposure to the test item and the determination of the permeability after the treatment provide an indication of the damaging effect of the test item.
For this purpose the induction of opacity and increased permeability in an isolated bovine cornea after application of the test item were measured.

2-Ethoxyethanol served as positive control. Saline was used as negative control.

Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
For equilibration and prior to application of the test item or controls, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130).


Permeability Determination
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer (software SoftMax Pro Enterprise, version 4.7.1).

DATA INTERPRETATION

Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:
IVIS In vitro Irritancy Score (according to OECD 437)
≤ 3 No Category (according to GHS)
> 3; < 55 No prediction can be made
≥ 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

99.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

In vivo

Irritant / corrosive response data:

Relative to the negative control, the test item Rewoteric QAM 50 caused a strong increase of the corneal opacity and the permeability (both higher than the ones caused by the positive control).

Any other information on results incl. tables

Results after 10 Minutes Incubation Time

Test Group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score
		Mean		Mean
Negative Control	0	0	0.050	0.051	0.75	0.76	Not categorized
	0		0.046		0.69
	0		0.056		0.84
Positive Control	54.00		0.636*		63.55	64.42	Category 1
	50.00		0.911*		63.67
	50.00		1.069*		66.04
Test substance	83.00		1.308*		102.63	99.33	Category 1
	70.00		1.608*		94.13
	83.00		1.216*		101.25

* corrected values

 

Applicant's summary and conclusion

Interpretation of results:

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

Remarks:

Migrated information

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, Rewoteric QAM 50 is seriously eye damaging.

Executive summary:

This in vitro study was performed to assess the corneal damage potential of the substance by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t130).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (0.9% (w/v) NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae.

Relative to the negative control, the test item caused a strong increase of the corneal opacity and the permeability. The calculated mean IVIS was 99.33 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 the test item is classified as seriously eye damaging.