1-Propanesulfonic acid, 3-(cyclohexylamino)-2-hydroxy-, sodium salt (1:1)

Endpoint summary

Description of key information

The test item induced neither a skin irritating nor a corrosive effect when tested in an EpiSkin model according to OECD TG 439 and TG 431, respectively (reference 7.3.1 -1 and 7.3.1 -2). In an in vitro eye irritation study according to OECD TG 437 the test item was identified as a corrosive or severe irritant (reference 7.3.2 -1).

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:

weight of evidence

Study period:

20 March 2013 to 09 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP

Version / remarks:

Version 1.8 (February 2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: reconstructed epidermis units

Details on animal used as source of test system:

not applicable

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkin SM)
- Tissue batch number(s): 13-EKIN-010
- Expiry date: 25 March 2013
- Date of initiation of testing: 20 March 2013

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: once
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: yes
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: Three replicates were used for the test item and controls respectively.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
According to the results of a previously performed study (Study no: 731.554.3856) the test item did not interact with the MTT and showed no ability to become coloured in contact with water, therefore using of additional controls were not necessary.

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

PREDICTION MODEL / DECISION CRITERIA
The test substance is considered to be irritant to skin (H315), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg

NEGATIVE CONTROL
- Amount applied: 20 μL
- Concentration: 1x PBS

POSITIVE CONTROL
- Amount applied: 20 μL
- Concentration: 5 % aq

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

1

Value:

103

Negative controls validity:

valid

Remarks:

100 %; SD 5.23

Positive controls validity:

valid

Remarks:

14 %; SD 1.75

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Cell viability

Substance	Optical Density (OD)		Viability (%)
Negative Control:1x PBS	1	0.661	100
	2	0.623	95
	3	0.691	105
	mean	0.658	100
	standard deviation (SD)		5.23
Positive Control:SDS (5 % aq.)	1	0.078	12
	2	0.101	15
	3	0.088	13
	mean	0.089	14
	standard deviation (SD)		1.75
Test Item:	1	0.639	97
	2	0.738	112
	3	0.652	99
	mean	0.676	103
	standard deviation (SD)		8.14

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP in vitro skin irritation test in the EPISKIN model, the test item was not irritating.

Executive summary:

EpiSkinTMSM test of the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 22 July 2010. Disks of EPISKIN (three units / chemical) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and PBS treated epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. The test item is considered to be irritant to skin, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean value: 103 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

In this in vitro skin irritation test in the EPISKIN model with the test item the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12 September 2012 to 27 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test”

Version / remarks:

February 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Details on animal used as source of test system:

not applicable

Justification for test system used:

The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM)
- Tissue batch number: 12-EKIN-033
- Expiry date: 17 September 2012
- Date of initiation of testing: 12 September

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: once
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item showed no direct interaction with MTT and therefore using of additional control, i.e. using killed epidermis, was not necessary. In addition, the test item showed no ability to become coloured in contact with water and therefore using of additional control was not necessary.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin, category 1C if mean tissue viability is ≥ 35 % after 1 hour exposure and < 35 % after 4 hours exposure according to Regulation (EC) 1272/2008.
- The test substance is considered to be non corrosive to skin, if mean tissue viability is ≥ 35 % after 4 hours exposure according to UN GHS.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 20 mg

NEGATIVE CONTROL
- Amount applied: 50 µL
- Concentration: 9 g/L

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

4 hours

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

2 per test item, negative and positive control

Details on test animals or test system and environmental conditions:

not applicable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

82

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

3 %

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Cell viability

Substance	Optical Density (OD)		Viability (%)
Negative Control:NaCl (9 g/L saline)	1	1.000	97
	2	1.058	103
	mean	1.029	100
Positive Control:Glacial acetic acid	1	0.033	3
	2	0.026	3
	mean	0.029	3
Test Item:	1	0.748	73
	2	0.935	91
	mean	0.841	82

Interpretation of results:

GHS criteria not met

Conclusions:

In the in vitro EPISKIN model test with the test item the results indicated that the test item is not corrosive to skin.

Executive summary:

EpiSkin™SM test of the test item has been performed to predict its corrosive potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 431, 13 April 2004. Disks of EPISKIN (two units / chemical) were treated with the test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 82 %. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. In conclusion, in this in vitro EPISKIN model test with the test item the results indicated that the test item is not corrosive to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2012 to 07 March 2013

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 Ocular Corrosives and Severe Irritants)

Version / remarks:

07 September 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

09 December 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

not applicable

Vehicle:

other: 0.9% sodium chloride solution

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL/per eye
- Concentration: 20%

VEHICLE
- Amount applied:750 µL/per eye
- Concentration: 0.9 %

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

No post-treatment incubation

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Fresh bovine eyes were obtained from the slaughterhouse Muller Fleisch GmbH, Enzstr. 2.4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank's balanced salt solution (supplemented with 0.01 % streptomycin and 0.01 % penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 hour.
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32°C. On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1 % fetal calf serum (= complete MEM) and stored in a water bath at 32 °C ± 1 °C. The same was performed with the MEM with phenol red. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM without phenol red was filled. The holders were then incubated for one hour in the incubation chamber at 32 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the arrival of the corneas they were examined and only corneas which were free from defects were used.

NUMBER OF REPLICATES
Two experiments were performed; the first experiment was not valid, because the positive control was not in the range of the validity data. The raw data of this experiment will be archived together with the other raw data, but will not be reported. The second experiment was valid. For each treatment group (negative control, positive control and test item), three replicates were used.

SOLVENT CONTROL USED
0.9 % sodium chloride solution

POSITIVE CONTROL USED
20 % imidazole solution

APPLICATION DOSE AND EXPOSURE TIME : 750 µL, 4 hours at 32°C

TREATMENT METHOD: Open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity was calculated from the measured absorption at 570 nm. The difference between opacity before and after exposition is used for further evaluation.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490) .

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
According to OECD Guideline no. 437 (2009) and EU method B.47, a substance that induces an IVIS > 55.1 is defined as a corrosive or severe irritant. Substances with IVIS < 55.1 may be considered as non-corrosive resp. not severely irritant.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

100.839

Vehicle controls validity:

valid

Remarks:

IVIS 1.458

Positive controls validity:

valid

Remarks:

IVIS 68.763

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, in both experiments
- Acceptance criteria met for positive control: yes, in the second experiment only

Table 1: In vitro irritation score (IVIS)

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative control (0.9 % NaCl)	1.447	1.458	0.7 %
	1.463
	1.464
Test item:	90.021	100.839	9.7 %
	103.615
	108.880
Positive control (20 % Imidazole)	75.187	68.763	8.1 %
	66.091
	65.010

 

Table 2: Absorption and Opacity Values

Parameter	Negative control			Positive control			Test item
Absorption before exposition	0.3707	0.3678	0.3751	0.2007	0.1812	0.1432	0.4041	0.2615	0.3362
Absorption after exposition	0.5714	0.5784	0.5847	1.6052	1.4840	1.4825	1.4320	1.5996	1.6576
Opacity before exposition	2.3480	2.3324	2.3719	1.5874	1.5177	1.3906	2.5357	1.8260	2.1687
Opacity after exposition	3.7273	3.7879	3.8433	40.2903	30.4789	30.3739	27.0396	39.7741	45.4569
Opacity difference	1.3793	1.4555	1.4713	38.7028	28.9612	28.9833	24.5039	37.9481	43.2882

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In an in vitro eye irritation test according to OECD Test Guideline 437 (BCOP), the test item was severely eye irritating/corrosive.

Executive summary:

In an in vitro eye irritation test according to OECD Test Guideline 437 (BCOP), the corneal irritation and damage potential of the test item was investigated by quantitative measurements of changes in opacity and permeability in a bovine cornea. Two experiments were performed; the first experiment was not valid, because the positive control was not in the range of the validity data. The raw data of this experiment will be archived together with the other raw data, but will not be reported. The second experiment was valid. The test item was applied onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for one hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes/four hours at 32 ± 1 °C. After removal of the test item and two hours post-incubation, opacity and permeability values were measured. Physiological sodium chloride solution was used as negative control, imidazole (20 % solution in 0.9 % sodium chloride solution) was used as positive control. The positive control induced a severe irritation on the cornea, mean IVIS was 68.763. The negative control showed no irritation, mean IVIS was 1.458. The test item was tested as 20 % solution. A mean IVIS of 100.839 was calculated, corresponding to an ICCVAM classification as "very severely eye irritant". According to OECD Guideline no. 437 (2009) and EU method B.47, a substance that induces an IVIS 55.1 is defined as a corrosive or severe irritant. No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

EpiSkin™SM test of the test item has been performed to predict its corrosive potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 431, 13 April 2004 (reference 7.3.1 -1). Disks of EPISKIN (two units / chemical) were treated with the test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 82 %. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. In conclusion, in this in vitro EPISKIN model test with the test item the results indicated that the test item is not corrosive to skin.

 

EpiSkin™SM test of the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 22 July 2010 (reference 7.3.1 -2). Disks of EPISKIN (three units / chemical) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and PBS treated epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. The test item is considered to be irritant to skin, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean value: 103 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

In this in vitro skin irritation test in the EPISKIN model with test item the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].

 

Eye irritation

In an in vitro eye irritation test according to OECD Test Guideline 437 (BCOP), the corneal irritation and damage potential of the test item was investigated by quantitative measurements of changes in opacity and permeability in a bovine cornea (reference 7.3.2 -1). Two experiments were performed; the first experiment was not valid, because the positive control was not in the range of the validity data. The raw data of this experiment will be archived together with the other raw data, but will not be reported. The second experiment was valid. The test item was applied onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for one hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes/four hours at 32 ± 1 °C. After removal of the test item and two hours post-incubation, opacity and permeability values were measured. Physiological sodium chloride solution was used as negative control, imidazole (20 % solution in 0.9 % sodium chloride solution) was used as positive control. The positive control induced a severe irritation on the cornea, mean IVIS was 68.763. The negative control showed no irritation, mean IVIS was 1.458. The test item was tested as 20 % solution. A mean IVIS of 100.839 was calculated, corresponding to an ICCVAM classification as "very severely eye irritant". According to OECD Guideline no. 437 (2009) and EU method B.47, a substance that induces an IVIS 55.1 is defined as a corrosive or severe irritant. No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data for skin and eye irritation/corrosion are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item is classified for eye damage, Cat.1 (H318) according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521. The test item is not to be classified for skin irritation/ corrosion according to the above mentioned Regulation.