1-Propanaminium, 3-amino-N-ethyl-N,N-dimethyl-, N-rape-oil acyl derivs., Et sulfates

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 July 14, 2017 to August 25, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008 of 30 May 2008

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-Propanaminium, 3-amino-N-ethyl-N,N-dimethyl-, N-rape-oil acyl derivs., Et sulfates

EC Number:

305-488-1

EC Name:

1-Propanaminium, 3-amino-N-ethyl-N,N-dimethyl-, N-rape-oil acyl derivs., Et sulfates

Cas Number:

94552-41-7

Molecular formula:

Molecular formula of the major constituents: C31H64N2O5S1 (C22:1 carbon chain) C27H56N2O5S1 (C18:1 carbon chain)

IUPAC Name:

ethyldimethyl{3-[(9Z)-octadec-9-enamido]propyl}azanium {3-[(13Z)-docos-13-enamido]propyl}(ethyl)dimethylazanium diethyl sulfate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier: MatTek
Date received: 22 August 2017
EpiDermTM Tissues (0.63cm2) lot number: 25838
Assay Medium lot number: D81717TMC
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL undiluted test substance

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

37°C, 5% CO2 for 3 h

Number of replicates:

Two tissue replicates for each treatment group

Test system

Vehicle:

unchanged (no vehicle)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

Exposure: 3 minutes

Run / experiment:

Test substance

Value:

ca. 86.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

Exposure: 60 minutes

Run / experiment:

Test substance

Value:

ca. 38.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Results 3

Irritation / corrosion parameter:

% tissue viability

Remarks:

Exposure: 3 and 60 minutes

Run / experiment:

Negative control

Value:

ca. 100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Results 4

Irritation / corrosion parameter:

% tissue viability

Remarks:

Exposure: 3 minutes

Run / experiment:

Positive control

Value:

ca. 3.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Corrosive

Results 5

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control

Value:

ca. 3.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Corrosive

Any other information on results incl. tables

Results

Direct MTT Reduction

An assessment found the test substance was able to directly reduce MTT. Therefore, an additional procedure using freeze killed tissues was performed. The results of the freeze killed tissues were subtracted from the mean OD of the test substance treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

 

Assessment of Color Interference with the MTT endpoint

The solution containing the test substance did not become colored. This was taken to indicate the test substance did not have the potential to cause color interference.

Test Substance, Positive Control Substance and Negative Control Substance

 

Mean OD₅₇₀values and viabilities for the negative control, positive control and test substance

Tissue	Exposure Period	Mean OD570of individual tissues (tvt)	Mean OD570of duplicate tissues	Corrected OD570(tvt- (tkt – ukt))	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.566	1.497	-	0.098	6.5	100*
		1.428
	60 Minutes	1.545	1.633	-	0.124	7.6
		1.720
Positive Control	3 Minutes	0.062	0.059	-	0.004	na	3.9
		0.056
	60 Minutes	0.058	0.058	-	0.001	na	3.5
		0.057
Test Substance	3 Minutes	1.268	1.319	1.297	0.071	5.4	86.6
		1.369
	60 Minutes	0.641	0.675	0.625	0.048	7.1	38.3
		0.709

tvt = Treated viable tissue

tkt = Treated killed tissue

ukt = Untreated killed tissue

na = Not applicable

 

3 minute exposure corrected mean OD₅₇₀=        0.139   (tkt) -  0.117   (ukt) = 0.022

60 minute exposure corrected mean OD₅₇₀=      0.148   (tkt) -  0.098   (ukt) = 0.050

 

Relative mean viability (%) = mean OD570 of test substance / mean of OD570 of negative control × 100

Coefficient of variation = standard deviation / mean OD570 of duplicate tissues × 100

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

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Substance
3 minute	100*	3.9	86.6
60 minute	100*	3.5	38.3

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

 

Quality Criteria

The initial test was repeated due to a failure to meet the assay acceptance criteria. The mean OD₅₇₀ for the negative control treated tissues was 1.497 for the 3‑Minute exposure period and 1.633 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 3.5% 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.

 

Conclusion

The test substance was considered to be non-corrosive to the skin.

Applicant's summary and conclusion

Interpretation of results:

other: non corrosive based on EU CLP criteria

Conclusions:

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

Executive summary:

An in vitro study was conducted to determine the corrosivity potential of the test substance, 'C18-unsatd. and C22-unsatd. AAP EDM-ES' (active: 104%), using the EpiDerm™ Human Skin Model, according to the OECD Guideline 431 and EU Method B.40 bis, in compliance with GLP. Duplicate tissues were treated with the 50 µL undiluted test substance and reference substances for exposure periods of 3 and 60 minutes. The test substance was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test substance was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT loading each tissue was placed in 2 mL 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 were presented in the form of percentage viability (MTT reduction in the test substance treated tissues relative to negative control tissues). Percentage viability for the test substance at 3 and 60 minutes were 86.6% and 38.3% respectively. The quality criteria required for acceptance of results in the test were satisfied. Under study conditions, the test substance was considered to be non-corrosive to the skin (Envigo, 2017).