4-(cyclohexylamino)butane-1-sulfonic acid

Endpoint summary

Description of key information

An in vitro skin corrosion test was conducted according to OECD 431 guideline and in accordance with GLP principles showed that the substance is not corrosive.

 

An in vitro skin irritation test, performed according to OECD guideline 439 and in accordance with GLP principles showed that no classification is required for skin irritation.

 

A EpiOcular™ test was performed according to OECD guideline 492 and in accordance with GLP principles showed that the test item is a non-irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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).

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

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 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

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.

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

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).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 April 2019 - 05 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142

Version / remarks:

31 May 2008.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue lot number: 303111 Kit F, keratinocyte strain 00267
- Surface: 0.6 cm^2
- Pretreatment: The tissues were kept on agarose and stored in the refrigerator on the day they were received. The next day, at least one hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 ml DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately one hour at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before the test item was applied.

INTERFERENCE WITH THE MTT ENDPOINT:
- Test for color interference by the test item: 25 mg test item was added to 50 μL Milli-Q water and the mixture was incubated for approx. one hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue/purple color change was observed. A negative control (Milli-Q water) was tested concurrently.
- Test for reduction of MTT by the test item: 25 mg of the test item was added to 50 μL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approx. one hour at 37.0 ± 1.0°C. At the end of the exposure time it was checked if a blue/purple color change was observed or a blue/purple precipitate was observed. A negative control (Milli-Q water) was tested concurrently.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment of 3 minutes: room temperature
- Temperature of 1 hour and post-treatment incubation: 36.8 - 37.9°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: after exposure and after incubation tissues were washed with phosphate buffered saline.
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Amount of MTT-medium: 300 μL
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: two tissues treated with test item + two replicates for the negative control and the positive control per exposure duration

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one experiment per exposure period (two in total) with 3 independent OD570 measurements per replicate.

ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
- The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

DECISION CRITERIA (see table 1 in 'any other information on materials and methods')
A test item is considered corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
- In addition, a test item considered non-corrosive (viability ≥50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
- In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 26.6 to 34.1 mg
- The test item was applied directly on top of the skin tissues

NEGATIVE CONTROL
- Amount applied: 50 μL (Milli-Q water)

POSITIVE CONTROL
- Amount applied: 50 μL (8N KOH)

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours in MTT medium

Number of replicates:

2 tissues per test item per exposure time (4 tissues in total)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

97

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

mean tissue viability (%): 100

Positive controls validity:

valid

Remarks:

mean tissue viability (%): 7.2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

87

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

mean tissue viability (%): 100

Positive controls validity:

valid

Remarks:

mean tissue viability (%): 9.8

Other effects / acceptance of results:

- RESULTS
Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, 4-(cyclohexylamino)butane-1-sulfonic acid is considered to be not corrosive. The mean tissue viability of the positive control was 7.2 and 9.8% after 3 minutes and one hour, respectively.

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
(see table 2 and 3 in 'any other information on results' for historical data and mean OD570 absorption data.):

- Acceptance criteria met for negative control:
yes, the absolute mean OD570 of the two tissues of the negative control was within the laboratory historical control data range (i.e 1.787 and 1.438 for the 3-minute and 1-hour exposure respectively).
- Acceptance criteria met for positive control:
yes, the mean relative tissue viability following 1-hour exposure to the positive control was <15 % (i.e. 9.8%).
- Acceptance criteria met for variability between replicate measurements:
yes, the Coefficient of Variation between tissue replicates was ≤30% for the negative control (3-minute: 0.0%; 1-hour: 4.1%) and test item (3-minute 0.2%; 1 hour: 1.9%).

Table 1 Decision criteria

Viability measured after 3-minutes and 1 hour	Prediction to be considered
Step 1
< 50% after 3 minute exposure	Corrosive
≥ 50% after 3 minute exposure AND < 15% after 1 hour exposure	Corrosive
≥ 50% after 3 minute exposure AND ≥ 15% after 1 hour exposure	Non-corrosive
Step 2 (for substances/mixtures identified as Corrosive in step 1)
< 25% after 3 minute exposure	Optional Sub-category 1A
≥ 25% after 3 minute exposure	A combination of optional Sub-categories 1B and 1C

 

Table 2 Historical control data

	Negative control		Positive control
	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (OD570)	1-hour treatment (OD570)
Range	1.258 – 2.615	1.371 – 2.371	0.092 – 0.56	0.046 – 0.339
Mean	1.73	1.78	0.19	0.14
SD	0.24	0.21	0.09	0.05
n	116	119	114	112

SD = Standard deviation n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2015 to November 2018.

 

Table 3 Mean Absorption in the in vitro Skin Corrosion Test with 4-(cyclohexylamino)butane-1-sulfonic acid

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.835	1.835	1.835	±	0.000	1.686	1.617	1.651	±	0.048
Test item	1.789	1.785	1.787	±	0.003	1.452	1.424	1.438	±	0.020
Positive control	0.126	0.137	0.132	±	0.007	0.201	0.121	0.161	±	0.056

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0471).  Isopropanol was used to measure the background absorption.

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin corrosion test performed according to OECD 431 and in accordance with GLP principles, 4-(cyclohexylamino)butane-1-sulfonic acid is found not corrosive to skin.

Executive summary:

In an in vitro skin corrosion test using a human skin model ( EpiDerm Skin Model) performed according to OECD 431 and in accordance with GLP principles, the ability of the test substance to induce skin corrosion on a human three dimensional epidermal model. The test substance (26.6 to 34.1 mg) was applied directly on top of 0.6 cm² cultured skin. After 3-minute and 1-hour treatments the substance was removed and the viability of the cells was tested by reduction of MTT. Viability of unexposed skin was set at 100%. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with test item compared to the negative control tissues was 97% and 87%, respectively. No color interference or direct reduction by MTT was observed in the test system. The positive and negative control were in historical control data range and were valid indicating that the test system functioned properly. Since the mean relative tissue viability was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, it can be concluded that the 4-(cyclohexylamino)butane-1-sulfonic acid is not corrosive in the in vitro skin corrosion test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23 April 2019 - 24 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse Vitelco, 's-Hertogenbosch, The Netherlands.
- Storage, temperature, and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL per cornea.
- Concentration: 20% (w/v) solution of test item

Duration of treatment / exposure:

240 ± 10 minutes at 32 ± 1°C

Duration of post- treatment incubation (in vitro):

90 ± 5 minutes in sodium-fluorescein for permeability determinations at 32 ± 1°C.

Number of animals or in vitro replicates:

3 replicates for the negative, positive, and treatment group each.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
The isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Fetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C The corneas were incubated for the minimum of 1 hour at 32 ± 1°C

QUALITY CHECK OF THE ISOLATED CORNEAS:
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 l of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) solution of the test item was introduced onto the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea.
Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies).
Possible pH effects of the test item on the corneas were recorded.
- POST-EXPOSURE INCUBATION:
90 ± 5 minutes in 5 mg Na-fluorescein/mL cMEM solution for permeability determinations

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: optical density at was measured in triplicate with the aid of microplate reader (TECAN Infinite® M200 Pro Plate Reader). OD490 values of less than 1.500 were used in the permeability calculation.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

DECISION CRITERIA: (see attached below):
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces IVIS >55 is defined as a corrosive or severe irritant (UN GHS: category 1);
For a test substance that induces an IVIS >3 and ≤ 55, no prediction on irritant potency can be made.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3 replicates

Value:

4.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean IVIS: 1.7

Positive controls validity:

valid

Remarks:

Mean IVIS: 166

Other effects / acceptance of results:

The test item induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 4.1 after 240 minutes of treatment
- The corneas treated with the test item showed opacity values of: 6.0, 4.4, 4.2 (corrected for negative control)
- Permeability values were: -0.011, -0.013, -0.008 (corrected for negative control)
- Individual IVIS scores were: 5.8, 4.2, 2.3

OTHER EFFECTS:
- The corneas were clear after the 240 minutes of treatment with the test item.
- No pH effect of the test item was observed on the rinsing medium

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, results were within historical range (IVIS ranging from 0.9 to 2.2).
- Acceptance criteria met for positive control: yes, results were within historical range (IVIS ranging from 123 to 205).
- See table 2 for historical data

Table 2 Historical Control Data for the BCOP studies

		Negative control		Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-4.4 – 5.2	-0.011 - 0.081	-4.3 – 5.4	86.5 – 206
Mean	0.95	0.01	1.15	143.48
SD	1.80	0.01	1.86	27.48
n	147	147	147	150

SD = Standard deviation n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Mar 2016 to Mar 2019.

            

Interpretation of results:

study cannot be used for classification

Conclusions:

In a Bovine Corneal Opacity and Permeability test (BCOP), performed according to OECD guideline 437 and in accordance with GLP principles, 4-(cyclohexylamino)butane-1-sulfonic acid induced an IVIS > 3 ≤ 55, therefore no conclusion on the classification for eye irritation or serious eye damage can be made.

Executive summary:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed with 4-(cyclohexylamino)butane-1-sulfonic acid according to OECD guideline 437 and in accordance with GLP principles. 750 µL of a 20% (w/v) solution of 4-(cyclohexylamino)butane-1-sulfonic acid was applied to corneas (n=3). The mean in vitro irritancy score of the positive control ( 20% (w/v) Imidazole) was 166, and the mean in vitro irritancy score of the negative control (physiological saline) was 1.7, which were both within historical range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

4-(cyclohexylamino)Butane-1-sulfonic acid did induce ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 4.1 after 240 minutes of treatment. Since 4-(cyclohexylamino)butane-1-sulfonic acid induced an IVIS > 3 ≤ 55, no conclusion on the classification for eye irritation or serious eye damage can be made.

Reference 2

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

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

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.

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In an in vitro skin corrosion test using a human skin model (EpiDerm Skin Model) performed according to OECD 431 and in accordance with GLP principles, the ability of the test substance to induce skin corrosion on a human three dimensional epidermal model was investigated. The test substance (26.6 to 34.1 mg) was applied directly on top of 0.6 cm² cultured skin. After 3-minute and 1-hour treatments the substance was removed and the viability of the cells was tested by reduction of MTT. Viability of unexposed skin was set at 100%. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with test item compared to the negative control tissues was 97% and 87%, respectively. No color interference or direct reduction by MTT was observed in the test system. The positive and negative control were in historical control data range and were valid indicating that the test system functioned properly.

 

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.

 

 

A Bovine Corneal Opacity and Permeability test (BCOP) was performed with 4-(cyclohexylamino)butane-1-sulfonic acid according to OECD guideline 437 and in accordance with GLP principles. 750 µL of a20% (w/v) solution of 4-(cyclohexylamino)butane-1-sulfonic acidwas applied to corneas (n=3). The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 166, and the mean in vitro irritancy score of the negative control (physiological saline) was 1.7, which were both within historical range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

4-(cyclohexylamino)Butane-1-sulfonic acid did induce ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 4.1 after 240 minutes of treatment. Since 4-(cyclohexylamino)butane-1-sulfonic acid induced an IVIS > 3 ≤ 55, no conclusion on the classification for eye irritation or serious eye damage can be made.

 

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%.

Justification for classification or non-classification

The available data shows that 4-(cyclohexylamino)butane-1-sulfonic acid is not corrosive and not irritating to the skin and therefore does not meet the criteria for classification according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

 

The available data shows that 4-(cyclohexylamino)butane-1-sulfonic acid is not an eye irritant and therefore does not need classification for eye irritation or serious eye damage according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).