4-(cyclohexylamino)butane-1-sulfonic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April 2019 - 29 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

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 skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EPISKIN Small Model(TM)
- Surface: 0.38 cm^2
- Tissue batch number:
19-EKIN-017
- Expiration date: 29 April 2019

ENVIROMENTAL CONDITIONS FOR TEST SYSTEM
- Temperature used during exposure to the test item:
Room temperature
- Temperature of incubations:
36.3 - 37.0°C containing 5.0 ± 0.5% CO2
- Humidity: 42 - 93%

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER EXPOSURE
- MTT concentration:
0.3 mg/mL in PBS
- Incubation time:
3 hours
- Spectrophotometer:
TECAN Infinite® M200 Pro Plate Reader.
- Wavelength:
570nm

NUMBER OF REPLICATE TISSUES:
3 tissues each for the test item, negative, and positive control.

ACCEPTABILITY CRITERIA

The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 single run


PREDICTION MODEL / DECISION CRITERIA (see table 1 in "any other information on materials and methods incl. tables")

A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.

A test item is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amounts applied: between 12.3 and 18.5 mg per tissue
- The skin tissue was moistened with 5 μL Milli-Q water before application.

NEGATIVE CONTROL
- Amount applied: 25 μL PBS per tissue

POSITIVE CONTROL
- Amount applied: 25 μL 5%(aq) SDS per tissue
- Positive control was re-spread after 7 minutes contact time

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours; + 3 hours with MTT

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates

Value:

108

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

mean tissue viability (%): 100

Positive controls validity:

valid

Remarks:

mean tissue viability (%): 13

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no (a check for Direct-MTT reduction was performed in Skin corrosion test using EpiDerm (Study No. 20191845).
- Colour interference with MTT: no (a check for colour interference was performed in Skin corrosion test using EpiDerm (Study No. 20191845).

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 and had a Standard Deviation value (SD) of 8.1 (% tissue viability).
- Acceptance criteria met for positive control: The positive control had a mean tissue viability after 15 ± 0.5 minutes exposure of 13% with a SD of 0.4 (% tissue viability).
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was < 10%.

- Since the mean relative tissue viability for 4-(cyclohexylamino)butane-1-sulfonic acid was above 50% the substanceis considered to be non-irritant.

Any other information on results incl. tables

In a previously performed skin corrosion study, the test item was found to be non-corrosive to the skin.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin irritation test, performed according to OECD guideline 439 and in accordance with GLP principles, 4-(cyclohexylamino)butane-1-sulfonic acid showed to be a non-irritant.

Executive summary:

In an in vitro skin irritation test, performed according to OECD guideline 439 and in accordance with GLP principles, the ability of 4-(cyclohexylamino)butane-1-sulfonic acid to induce skin irritation was tested using reconstructed human epidermis (EPISKIN-SM). The test substance was applied to 0.38 cm² cultured skin. After 15 minutes exposure, the substance was removed and the tissues were incubated for 42 hours. Survival of unexposed tissue (negative control) was set at 100%. The positive control had a mean tissue viability of 13%. The test substance showed a mean tissue viability of 108% compared to the negative control. Since the mean relative tissue viability after exposure to the test substance was above 50%, no classification is required for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).