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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 23, 2017 to September 04, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 23, 2017 to September 04, 2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 29 August 2017
EpiSkinTM Tissues (0.38cm2) lot number : 17-EKIN-035
Maintenance Medium lot number : 17-MAIN3-037
Assay Medium lot number : 17-ESSC-034

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 μL (26.3 μL/cm2)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

37ºC, 5% CO2 in air for 42 h

Number of replicates:

3 (Three)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test substance

Value:

ca. 113.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean tissue viability for the positive control treated tissues was 6.6% relative to the negative control treated tissues and the standard deviation value of the viability was 2.9%. The positive control acceptance criteria were therefore satisfied. The mean OD570 for the negative control treated tissues was 0.807 and the standard deviation value of the viability was 2.5%. The negative control acceptance criteria were therefore satisfied. The standard deviation calculated from individual tissue viabilities of the three identically test substance treated tissues was 1.4%. The test substance acceptance criterion was therefore satisfied.

Results

 

Direct MTT reduction

The MTT solution containing the test substance did not turn blue or purple which indicated that the test substance did not directly reduce MTT.

 

Assessment of color interference with the MTT endpoint

The solution containing the test substance was a very pale green color. This color was attributed to the intrinsic color of the test substance itself. It was therefore unnecessary to run color correction tissues.

 

Test substance, positive control substance and negative control substance

The individual and mean OD₅₇₀values, standard deviations and tissue viabilities for the test substance, negative control substance and positive control substance are given in table 1. The mean viabilities and standard deviations of the test substance and positive control, relative to the negative control are also given in table 1.

 

Table 1: Mean OD₅₇₀ values and viabilities for the negative control substance, positive control substance and test substance

Substance	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Substance	0.794	0.807	0.020	98.4	100	2.5
	0.830			102.9
	0.797			98.8
Positive Control Substance	0.078	0.053	0.024	9.7	6.6	2.9
	0.031			3.8
	0.051			6.3
Test Substance	0.928	0.917	0.011	115.0	113.6	1.4
	0.906			112.3
	0.916			113.5

The relative mean viability of the test substance treated tissues was113.6% after a 15‑Minute exposure period and 42‑h post‑exposure incubation period. It was considered unnecessary to perform IL-1aanalysis as the results of the MTT test were unequivocal.

Conclusion

The test substance was classified as non-irritant. The following classification criteria apply:

EU CLP not classified for irritation.

UN GHS not classified for irritation (category 3 cannot be determined).

Interpretation of results:

other: Not classified based on EU CLP Criteria

Conclusions:

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

Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'C18-unsatd and C22-unsatd. AAP EDM-ES' (active: 104%), using EPISKIN TM reconstructed human epidermis model, according to the OECD Guideline 439 and EU Method B46, in compliance with GLP. Triplicate tissues were treated with the test substance (undiluted) for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 h. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96 well plate. The optical density was measured at 570 nm. Results were presented in the form of percentage viability (MTT reduction in the test substance treated tissues relative to negative control tissues). The relative mean viability of the test substance treated tissues was determined to be 113.6% after the 15 minute exposure period and 42 h post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. Under study conditions, the test substance was considered to be non-irritating to the skin (Envigo, 2017).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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-lanolin acyl derivs., Et sulfates

EC Number:

276-339-5

EC Name:

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

Cas Number:

72102-40-0

Molecular formula:

C29H62N2O5S (iC20 - representative)

IUPAC Name:

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

Test material form:

solid

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:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 29 August 2017
EpiSkinTM Tissues (0.38cm2) lot number : 17-EKIN-035
Maintenance Medium lot number : 17-MAIN3-037
Assay Medium lot number : 17-ESSC-034

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 μL (26.3 μL/cm2)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

37ºC, 5% CO2 in air for 42 h

Number of replicates:

3 (Three)

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test substance

Value:

ca. 113.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean tissue viability for the positive control treated tissues was 6.6% relative to the negative control treated tissues and the standard deviation value of the viability was 2.9%. The positive control acceptance criteria were therefore satisfied. The mean OD570 for the negative control treated tissues was 0.807 and the standard deviation value of the viability was 2.5%. The negative control acceptance criteria were therefore satisfied. The standard deviation calculated from individual tissue viabilities of the three identically test substance treated tissues was 1.4%. The test substance acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Results

 

Direct MTT reduction

The MTT solution containing the test substance did not turn blue or purple which indicated that the test substance did not directly reduce MTT.

 

Assessment of color interference with the MTT endpoint

The solution containing the test substance was a very pale green color. This color was attributed to the intrinsic color of the test substance itself. It was therefore unnecessary to run color correction tissues.

 

Test substance, positive control substance and negative control substance

The individual and mean OD₅₇₀values, standard deviations and tissue viabilities for the test substance, negative control substance and positive control substance are given in table 1. The mean viabilities and standard deviations of the test substance and positive control, relative to the negative control are also given in table 1.

 

Table 1: Mean OD₅₇₀ values and viabilities for the negative control substance, positive control substance and test substance

Substance	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Substance	0.794	0.807	0.020	98.4	100	2.5
	0.830			102.9
	0.797			98.8
Positive Control Substance	0.078	0.053	0.024	9.7	6.6	2.9
	0.031			3.8
	0.051			6.3
Test Substance	0.928	0.917	0.011	115.0	113.6	1.4
	0.906			112.3
	0.916			113.5

The relative mean viability of the test substance treated tissues was113.6% after a 15‑Minute exposure period and 42‑h post‑exposure incubation period. It was considered unnecessary to perform IL-1aanalysis as the results of the MTT test were unequivocal.

Conclusion

The test substance was classified as non-irritant. The following classification criteria apply:

EU CLP not classified for irritation.

UN GHS not classified for irritation (category 3 cannot be determined).

Applicant's summary and conclusion

Interpretation of results:

other: Not classified based on EU CLP Criteria

Conclusions:

Based on the results of the read across study, the test substance, iso and anteiso C10-40 AAP EDM-ES, can be considered to be non-irritating to the skin.

Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the read across substance, 'C18-unsatd and C22-unsatd. AAP EDM-ES' (active: 104%), using EPISKIN TM reconstructed human epidermis model, according to the OECD Guideline 439 and EU Method B46, in compliance with GLP. Triplicate tissues were treated with the test substance (undiluted) for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 h. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96 well plate. The optical density was measured at 570 nm. Results were presented in the form of percentage viability (MTT reduction in the test substance treated tissues relative to negative control tissues). The relative mean viability of the test substance treated tissues was determined to be 113.6% after the 15 minute exposure period and 42 h post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. Under study conditions, the test substance was considered to be non-irritant to the skin (Envigo, 2017). Based on the results of the read across study, the test substance, 'iso and anteiso C10-40 AAP EDM-ES' can be considered to be non-irritating to the skin.

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