4-(cyclohexylamino)butane-1-sulfonic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 July 2019 - 12 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

June 25, 2018

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-(cyclohexylamino)butane-1-sulfonic acid

EC Number:

629-080-0

Cas Number:

161308-34-5

Molecular formula:

C10 H21 N O3 S

IUPAC Name:

4-(cyclohexylamino)butane-1-sulfonic acid

Test material form:

solid

Details on test material:

Physical Description: White solid
Storage Conditions: At room temperature

Test animals / tissue source

Species:

human

Strain:

other: normal human keratinocytes

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27487 Kit C). Source: MatTek Corporation, Ashland MA, U.S.A.

The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 55.1 to 78.2 mg

Duration of treatment / exposure:

6 hours ± 15 minutes

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes post incubation
180 ± 10 minutes for cell viability measurement

Number of animals or in vitro replicates:

2

Details on study design:

Test for Color Interference by the Test Item
To assess the color interference, approximately 50 mg of the test item or 50 μL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark. Furthermore, approximately 50 mg of the test item or 50 μL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 hours at room temperature
with gentle shaking. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g.
At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Test for Reduction of MTT by the Test Item
To assess the ability of the test item to reduce MTT, approximately 50 mg of the test item was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for 1 hour at 37.0 ± 1.0°C in the dark. A negative control, 50 μL sterile Milli-Q water was tested concurrently. If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT.

Test System Set Up:
On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for
20 ± 4 hours at 37°C in 1.0 mL fresh pre-warmed Assay Medium.

Application/Treatment of the Test Item:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 54 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.8 - 37.0°C).

Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free- DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
Two tissues were treated with 50 μL Milli-Q water (negative control) and 2 tissues with 50 μL Methyl Acetate (positive control) respectively. At least 50 mg solid was added into the 6-well plates on top of the tissues. After the exposure period with the test item (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item.
After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a prelabeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post- Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.

Cell Viability Measurement:
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium
(1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking.
The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Calculation of Cell Viability:
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows: %Viability = (ODc/mean ODlt_u+MTT) * 100

Acceptability criteria:
The in vitro eye irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.
c) The difference between the % tissue viabilities of the two identically treated replicates should be <20.

Interpretation:
The test chemical is identified as not requiring classification and labelling according to Regulation (EC) No. 1272/2008 and its amendments if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.

Results and discussion

In vitro

Results

Irritation parameter:

other: Mean tissue viability (%)

Run / experiment:

Mean of 2 experiments

Value:

87

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

29%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- Interference of the Test Item with the MTT Endpoint: Because no color changes were observed it was concluded that 4-(cyclohexylamino)butane-1-sulfonic acid did not interact with the MTT endpoint and it was concluded that the test item did not induce color interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control:
The positive control had a mean cell viability after 6 hours ± 15 minutes minutes exposure of 29%.
- The difference between the percentage of viability of two tissues treated identically was less than 17%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the outcome of an EpiOcular™ test performed according to OECD 492 guideline and GLP principles, it is concluded that 4-(cyclohexylamino)butane-1-sulfonic acid is not irritating to the eye.

Executive summary:

An EpiOcular™ test was performed with 4-(cyclohexylamino)butane-1-sulfonic acid according to OECD guideline 492 and in accordance with GLP principles. The mean cell viability of the positive control was 29%, and the mean cell viability of the negative control 100%. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with 4-(cyclohexylamino)butane-1-sulfonic acid compared to the negative control tissues was 87%. Since the mean relative tissue viability for 4-(cyclohexylamino)butane-1-sulfonic acid was above 60% after 6 hours ± 15 minutes treatment, 4-(cyclohexylamino)butane-1-sulfonic acid is considered to be non-irritant to the eye and does not need to be classified according to Regulation (EC) No. 1272/2008 and its amendments.