[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 April 2020 - 17 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine

IUPAC Name:

Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine

Test material form:

liquid

Details on test material:

Molecular weight: 357.269
Appearance: light amber viscous liquid
Storage conditions: room temperature, in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Justification for test system used:

According to OECD guideline 431

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Model used: EpiDerm Reconstucted Human Epidermis
Tissue batch number: 30867
CoA signed: 20 May 2020


TEMPERATURE USED FOR TEST SYSTEM
Temperature used during treatment / exposure: 37 °C, 5% CO2


REMOVAL OF TEST MATERIAL AND CONTROLS
Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
MTT concentration: A 1.0 mg/mL MTT solution was prepared from a MatTek MTT-100 kit immediately prior to usage.
Incubation time: 3-Minute and 60-Minute exposure periods
Spectrophotometer: Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader and LT-com analysis software.


NUMBER OF REPLICATE TISSUES:
Two replicate tissues



PREDICTION MODEL / DECISION CRITERIA
Viability Measured after Exposure Time Points: Prediction to be considered according to EU CLP Regulation (EC) No 1272/2008 UN GHS

STEP 1
< 50% after 3 min exposure : Corrosive
≥ 50% after 3 min exposure AND < 15% after 60 min exposure: Corrosive
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure: Non-corrosive

STEP 2 for test items identified as corrosive in step 1
< 25% after 3 min exposure: H314 (Sub-category 1A)
≥ 25% after 3 min exposure: H314 (Combination of sub-categories 1B-and-1C)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

For test item, positive and negative control: 50 µL applied

Duration of treatment / exposure:

3-Minute and 60-Minute exposure periods

Number of replicates:

Two replicate tissues for the test item, positive and negative control

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute (mean)

Value:

84.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute (mean)

Value:

62.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean OD570 for the negative control treated tissues was 2.005 for the 3-Minute exposure period and 2.062 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 2.9% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.

In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

The relative mean viabilities for each treatment group were as follows:

 

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	3.3	84.3
60 minute	100*	2.9	62.5

 

 

 

Mean OD570values and viabilities for the negative control, positive control and test item are given in Appendix 1 (appended to attached background material) and replicated in the table below:

 

Tissue	Exposure Period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 minutes	1.621	2.005	0.543	27.1	100*
		2.389
	60 minutes	2.030	2.062	0.045	2.2
		2.093
Positive Control	3 minutes	0.065	0.066	0.001	N/A	3.3
		0.066
	60 minutes	0.059	0.059	0.000	N/A	2.9
		0.059
Test Item	3 minutes	1.850	1.691	0.225	13.3	84.3
		1.532
	60 minutes	1.379	1.288	0.129	10.0	62.5
		1.197

Applicant's summary and conclusion

Interpretation of results:

other: Not classified according to EU criteria

Conclusions:

In this study and under the experimental conditions reported the test item was considered to be non-corrosive to the skin.

Executive summary:

Introduction

The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue.  Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control.  Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot.  The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

 

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period.  At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading.  After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction.  

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate.  The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Results

The relative mean viabilities for each treatment group were as follows:

 

Negative control

3 minutes - 100%*

60 minutes - 100%*

* The mean viability of the negative control tissues is set at 100%

 

Positive control

3 minutes - 3.3%

60 minutes - 2.9%

 

Test item

3 minutes - 84.3%

60 minutes - 62.5%

 

Acceptance criteria:  The criteria required for acceptance of results in the test were satisfied.

 

Conclusion

In this study and under the experimental conditions reported: The test item was considered to be non-corrosive to the skin.