Cyclohexanaminium, N,N,N-trimethyl-, sulfate (2:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD (2014a) Draft Proposal for a New Test Guideline: Reconstructed Human Cornealike Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Trimethylcyclohexylammonium sulfate

IUPAC Name:

Trimethylcyclohexylammonium sulfate

Constituent 2

Reference substance name:

bis(N,N,N-trimethylcyclohexanaminium) sulfate

EC Number:

807-085-0

Cas Number:

1004297-30-6

Molecular formula:

2 C9 H20 N .O 4S

IUPAC Name:

bis(N,N,N-trimethylcyclohexanaminium) sulfate

Details on test material:

- Name of test material (as cited in study report): 1004297-30-6
- Physical state: Solid / yellowish
- Analytical purity: 95.9 wt.%
- Lot/batch No.: VUONGH-00104

Test animals / tissue source

Species:

other: in vitro

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

a bulk volume of 50 μL (about 24 mg) of the test material was applied covering the whole tissue surface.

Details on study design:

Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest was performed. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.
In case where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control, in the same way as described in the following section.
Basic procedure
Several test substances were tested in parallel within the present test (test no. 59) using the same control tissues (NC and PC). Two tissues were treated with each, the test substance, the PC and the NC. There are two separate protocols for liquids and solids, differing in exposure time and postincubation
period. Due to the physical condition of the test substance the protocol for solids was applied.
Pre-incubation of the tissues
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.
Pretreatment of the tissues
After the pre-incubation the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
Application of the test substance
Using a sharp spoon, a bulk volume of 50 μL of the test material was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.
Removal of the test substance and postincubation period
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance. After 25 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 18 hours (postincubation period).
MTT incubation
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

Results and discussion

Any other information on results incl. tables

Test substance		tissue 1	tissue 2	mean	inter-tissue variability [%]
NC	mean OD570	1.709	1.691	1.700
	viability [% of NC]	100.5	99.5	100.0	1.1
14/0711-1	mean OD570	0.378	0.268	0.323
	viability [% of NC]	22.3	15.8	19.0	6.5
PC	mean OD570	0.427	0.550	0.489
	viability [% of NC]	25.1	32.4	28.7	7.3

Based on the observed results and applying the evaluation criteria, it was concluded, that Trimethylcyclohexylammonium sulfate shows an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.