Benzenemethanaminium, N,N,N-trimethyl-, 2-hydroxy-2-methylpropanoate (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 April 2012 to 02 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD TG No. 437, September 07, 2009 “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: Commission Regulation (EU) No. 1152/2010 of 8 December 2010 Part B: Bovine Corneal Opacity and Permeability Test, Method for identifying ocular corrosives and severe irritants; Official Journal of the European Union, No. L324

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

benzyltrimethylazanium 2-hydroxy-2-methylpropanoate

EC Number:

805-580-6

Cas Number:

1431696-36-4

Molecular formula:

C14 H23 O3 N

IUPAC Name:

benzyltrimethylazanium 2-hydroxy-2-methylpropanoate

Test material form:

other: solid

Details on test material:

- Purity: The test substance has been characterized analytically.
- Homogeneity: The test substance was homogeneous by visual inspection.
- Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- pH-value: Ca. 7 (undiluted test substance, moistened with water). Ca. 5 (20% aqueous preparation)
- Physical state: Solid

Test animals / tissue source

Species:

other: isolated bovine cornea

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The test system (target tissue) is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months). Supplier: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim, Germany

Test system

Vehicle:

other: de-ionized water

Amount / concentration applied:

20% (w/v) solution in de-ionized water

Duration of treatment / exposure:

4h

Observation period (in vivo):

Not applicable.

Number of animals or in vitro replicates:

3 corneas per treatment group

Details on study design:

- Preparation of the bovine corneas and measurement of initial corneal opacity: Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders that consists of anterior and posterior chambers. Both chambers were filled to excess with prewarmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.
After the equilibration period the medium in both chambers was replaced with fresh prewarmed
medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 507 opacity units1 were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups. Each corneal holder was uniquely identified with a number on the chambers.

- Application of the test substance and washing: Each treatment group (test substance, NC and PC) consisted of 3 corneas. Before application the medium in the anterior chamber was removed using a syringe. 750 μL of the 20% (w/v) test-substance preparation (non-surfactant) was applied into the
anterior chamber using a pipette. Control tissues were concurrently applied into the anterior chamber with 750 μL of de-ionized water (negative control, NC) or with 750 μL of 20% (w/v) solution of Imidazole in de-ionized water (positive control, PC) using a pipette. The corneas were incubated in a horizontal position at about 32 °C for approximately 4 hours (non-surfactant solids). The test substance, NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).

- Measurement of final corneal opacity: Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.

- Determination of permeability: For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (5 mg/mL for solid test substances) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: Opacity score

Basis:

mean

Time point:

other: 4h

Score:

28

Reversibility:

other: not applicable

Results 2

Irritation parameter:

other: Permeability score

Basis:

mean

Time point:

other: 4h

Score:

0.006

Reversibility:

other: not applicable

Results 3

Irritation parameter:

other: in vitro irritancy score (IVIS)

Basis:

mean

Time point:

other: 4h

Score:

28

Reversibility:

other: not applicable

Remarks on result:

other: A score of 55 or higher indicates highly irritatig properties.

Irritant / corrosive response data:

Based on the observed results and applying the evaluation criteria it was concluded, that the substance does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.

Applicant's summary and conclusion

Interpretation of results:

other: not highly irritating

Remarks:

Criteria used for interpretation of results: EU