Protein hydrolyzates, silk, reaction products with 3-chloro-2-hydroxypropyl-cocoalkyl-dimethylammonium chloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 24, 2018 to February 01, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

{3-[(1-carboxy-2-hydroxyethyl)amino]-2-hydroxypropyl}(dodecyl)dimethylazanium {3-[(1-carboxyethyl)amino]-2-hydroxypropyl}(dodecyl)dimethylazanium {3-[(carboxymethyl)amino]-2-hydroxypropyl}(dodecyl)dimethylazanium trichloride

EC Number:

947-925-4

Molecular formula:

Molecular formula of major active constituents: C20H43CL1N2O3 (representative molecular formula for C12 alkyl chain Quaternised alanine) C19H41CL1N2O3 (representative molecular formula for C12 alkyl chain Quaternised glycine) C20H43Cl1N2O4 (representative molecular formula of C12 alkyl chain quaternised serine)

IUPAC Name:

{3-[(1-carboxy-2-hydroxyethyl)amino]-2-hydroxypropyl}(dodecyl)dimethylazanium {3-[(1-carboxyethyl)amino]-2-hydroxypropyl}(dodecyl)dimethylazanium {3-[(carboxymethyl)amino]-2-hydroxypropyl}(dodecyl)dimethylazanium trichloride

Test material form:

other: Paste

In vitro test system

Test system:

human skin model

Remarks:

The reconstructed human epidermal model EpidermTM (EPI-200 MatTek Corporation)

Source species:

human

Cell type:

other: normal human-derived epidermal keratinocytes

Justification for test system used:

The EpiDermTM skin model and assay for skin corrosion testing is endorsed by OECD TG 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

Test system
The reconstructed human epidermal model EpidermTM (EPI-200 MatTek Corporation) consists of normal human-derived epidermal keratinocytes which have been cultured to form a multi-layered highly differential model of the human epidermis. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Characterisation of the test system
MatTek’s EpiDermTM model has been extensively characterised for multiple parameters including morphology, tissue viability, skin barrier function and sterility. QC results for the specific lot of models received (Lot# 25876) were checked in-house for MatTek acceptance ranges with the following outcome:
- Morphology - PASS
- Tissue viability - PASS
- Skin barrier function (ET50 value for 1 % Triton X-100) where ET50 is the time taken for 1 % Triton X-100 to reduce the viability of the skin model to 50 % relative to the negative control) - PASS
- Sterility testing showed no contamination during long term antibiotic and antimycotic free culture - PASS

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Single topical application of 15 μL sterile water plus neat test substance
50 μL reference substances

Duration of treatment / exposure:

3 and 60 minutes at 37°C, 5 % CO2, 95 % RH

Number of replicates:

3 replicates each for test substance, negative and positive controls

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

ca. 95.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

ca. 53.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Other effects / acceptance of results:

Prior to the assay, the test substance was checked for interference (water coloration or MTT interference) and found not to interfere.

Any other information on results incl. tables

Results

Table 1: Cell viability measurements after 3 minutes of application

Name	Tissue n°	3 min endpoint
		Aliq. 1	Aliq. 2	mean	OD Mean	viability	Mean	SD	CV
						[%]	[%]	[%]	[%]
NC	1	1.642	1.604	1.623	1.561	104.0	100.0	3.4	3.4
	2	1.536	1.516	1.526		97.7
	3	1.554	1.516	1.535		98.3
PC	1	0.374	0.356	0.365	0.379	23.4	24.3	0.8	3.3
	2	0.388	0.382	0.385		24.6
	3	0.390	0.385	0.387		24.8
TA2	1	1.496	1.459	1.477	1.486	94.6	95.2	0.6	0.6
	2	1.509	1.483	1.496		95.8
	3	1.478	1.491	1.484		95.1

NC: negative control (H₂O), PC: Positive control (KOH 8N), TA2: Test substance

Table 2: Cell viability measurements after 1 h of application

Name	Tissue n°	1h endpoint
		Aliq. 1	Aliq. 2	mean	OD Mean	viability	Mean	SD	CV
						[%]	[%]	[%]	[%]
NC	1	1.565	1.551	1.558	1.548	100.7	100.0	3.9	3.9
	2	1.492	1.473	1.483		95.8
	3	1.617	1.586	1.602		103.5
PC	1	0.062	0.052	0.057	0.048	3.7	3.1	0.7	21.7
	2	0.035	0.038	0.037		2.4
	3	0.047	0.054	0.051		3.3
TA2	1	0.969	0.954	0.962	0.830	62.1	53.6*	12.1	22.5
	2
	3	0.692	0.702	0.697		45.1

*corrected value in (one outlier removed for TA2, i.e., second tissue readings)

NC: negative control (H₂O), PC: Positive control (KOH 8N), TA2: Test substance

 

Table 3: Mean and SD of cell viability measurements after 3 minutes and 1 h application

	3 min			1 h
	Mean of viability [%]	SD of viability	CV(%)	Mean of viability [%]	SD of viability	CV(%)
NC	100.0	3.4	3.4	100.0	3.9	3.9
PC	24.3	0.8	3.3	3.1	0.7	21.7
TA2	95.2	0.6	0.6	53.6	12.1	22.5

NC: negative control (H₂O), PC: Positive control (KOH 8N), TA2: Test substance.

 

Table 4: Results Summary

Test substance	Test substance ID	Viability after 3 minutes application (% to negative control)	Viability ≥ 50% after 3 min (Yes/No)	Viability after 1h application (% to negative control)	Viability ≥ 15% after 1h (Yes/No)	Corrosive (C)/Non corrosive(NC)
Test substance	TA2	95 .2%	Yes	53.6%	Yes	NC

The test substance did not reduce the viability below 50% after 3 min nor below 15% after 1h and should be considered as non-corrosive. Note that the final results are presented here (without outliers).

Acceptance criteria

		Actual values	Pass/Failed
Acceptance criterion 1	The mean OD570of the negative control tissues must be ≥0.8.	3 min: 1.561 1h: 1.548	Pass
Acceptance criterion 2	The mean of the positive control relative percentage viability, after 1 hour exposuremust be < 15% of the mean of the negative control.	3.1%	Pass
Acceptance criterion 3	In the range between 20% and 100% viability, the coefficient of variation (CV) is an additional acceptance criterion.It should not exceed 0.3(i.e 30%).	CV values 3min 1h NC 3.4 3.9 PC 3.3 21.7 TA2 0.6 22.5	Pass

Interpretation of results and skin corrosion Prediction Model

 

The cut-off values for the prediction of human skin corrosion are as follows:

 

Step 1

A test substance is classified "corrosive", if the relative tissue viability after3 mintreatment with a test material is decreased below 50%.

In addition, those materials classified "non-corrosive" after3 min(viability ≥ 50%) are classified "corrosive" if the relative tissue viability after1 htreatment with a test material is decreased below 15%.

 

Mean tissue viability(expressed as % of negative control)	Prediction
3 min < 50%	corrosive
3 min ≥ 50%and1 h: < 15%	corrosive
3 min ≥ 50%and1 h: ≥ 15%	non-corrosive

 

Step 2 (if test substance is classified as corrosive in step 1)

A test substance is classified "corrosive, optional Sub-Category 1A", if the relative tissue viability after3 mintreatment with a test material is decreased below 25%.

A test substance is classified "corrosive, optional Sub-Category 1B/1C", if the relative tissue viability after3 mintreatment with a test material is ≥25%.

 

Mean tissue viability(expressed as % of negative control)	Prediction
3 min < 25%	Corrosive, optional Sub-category 1A
3 min ≥ 25%	Corrosive, optional Sub-categories 1B and 1C

Conclusion for test substance

Test substance evaluated for skin corrosion following OECD guideline TG 431 and using EpiDerm^TM tissue model was non-corrosive.

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive based on CLP criteria

Conclusions:

Under the study conditions, the test substance was determined to be non-corrosive to the skin.

Executive summary:

An in vitro study was conducted to determine the skin corrosion potential of the test substance, 'Cocodimonium hydroxypropyl hydrolysed silk' (active: 51%), using Reconstructed Human Epidermis (RHE) cells, according to OECD Guideline 431, in compliance with GLP. EpiDermTM tissues were kept overnight at 4°C. On Day 1, the tissues were pre-incubated for 1 h at 37°C, 5% CO2, 95% RH. After incubation, tissues were exposure to test (15 μL water plus neat test substance) and reference substances (50 μL sterile water as negative control and 50 μL Potassium hydroxide as positive control) in triplicates for 3 and 60 minutes. After 3 minutes and 1 h treatment, the test substance and the reference substances were rinsed off from the tissues. Cell viability of the tissues was evaluated by addition of MTT on Day 2 and final MTT assay testing and measurements were performed. Results were compared to negative control. All validity criteria for the performed test were met. After 3 minutes and 1 h treatment, the mean viability values obtained with the test substance were determined to be 95.2% and 53.6%, respectively, which is well above the corrosive limits of 50 and 15% respectively. Under the study conditions, the test substance was determined to be non-corrosive to the skin (XCellR8, 2018).