1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with C18-unsatd. fatty acids, Me sulfates (salts)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-11-24 to 2010-11-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(23 July 2009)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(22 July 2010)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C18 unsatd., Me sulfates (salts)

IUPAC Name:

1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C18 unsatd., Me sulfates (salts)

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material: 1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C18 unsatd., Me-sulfates (salts)
- Physical state: liquid
- Analytical purity: 100%

Test animals

Species:

other: in vitro test

Test system

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Controls:

other: positive, negative and quality control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL

Duration of treatment / exposure:

60 ± 1 min

Observation period:

42 ± 2 h

Number of animals:

triplicates

Details on study design:

TEST SYSTEM
EpiDerm EPI-200-SIT (MatTek Corporation, 200 Homer Avenue, Ashland, MA 01721, USA); Lot: 13885

APPLICATION
- test substance was applied topically for 60 min (± 1 min) to the EpiDerm RHE
- after washing: post incubation period of a total of 42 h (± 2 h) at 37°C (± 1°C) and 5% CO2 (± 1%)
- cell viability was determined by using the standard MTT assay

Negative control: 30 µL ultrapure water, 60 ± 1 min
Positive control: 30µL 5% SDS, 60 ± 1 min
Quality control: 30 µL 1% Triton X-100, 2 h ± 15 min

REMOVAL OF TEST SUBSTANCE
- after 60 min rinsing with PBS (15 times)

MTT ASSAY
- at the end of the 42 h (± 2 h) post incubation period, tissues were transferred into 24 well plate prepared for the Standard MTT Assay, each well containing 300 µL MTT medium
- the plates were incubated for 3 h (± 5 min) at 37°C (± 1°C) and 5 % CO2 (± 1 %)
- formazan dye was extracted from the tissues with Isopropanol for 2 h at room temperature while shaking at approx. 120 rpm
- from each tissue 2 x 200 µL aliquots of the blue formazan solution were transferred into a 96 well microtiter plate; OD was determined at 550 nm

DATA ANALYSIS
- viability in % of the treated tissues was calculated in relation to the corresponding negative control

SCORING SYSTEM:
- cell viability measurement (MTT assay, OD at 550 nm)
- mean tissue vialbility > 50% = non-irritant
- mean tissue viability

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

82.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Although residual test substance adhered to the reconstructed epidermis after the rinsing procedure, the overall performance of the study was not affected, because it has been demonstrated that the test substance did not chemically reduce MTT and the cell viability was not decreased below the defined threshold limits.
The positive control (5% SDS) decreased the cell viability to 7.8%. The quality control (1% Triton X-100) experiment resulted in a tissue viability of 95.9% demonstrating intact barrier function of the test system.

Any other information on results incl. tables

No direct MTT reduction capacity of the test substance has been observed.

 

Viability of epidermal models:

 

	Quality control - Skin Barrier 1% Triton X-100	Negative control	Positive control 5% SDS	Test substance
60 min exposure; 42 h post exposue	-	100.0 (± 1.2)	7.8 (± 1.3)	82.7 (± 1.5)
2 h exposure	95.5 (± 1.4)	100.0 (± 4.0)	-	-
result	valid	valid	valid	Not irritant

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

MDIPA Esterquat C18 unsatd. is not irritating in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

In a dermal irritation study according to OECD Guideline 439 (In Vitro Skin Irritation) (22 July 2010) and EU method B.46 (In vitro skin irritation: reconstructed human epidermis model test) (23 July 2009), 30 µL MDIPA Esterquat C18 unsatd. (100% a.i.) was applied in triplicates for 60 min to a three-dimensional human epidermis model (EpiDerm EPI-200-SIT, MatTek Corporation).

After 60 minutes exposure at 37°C, the tissues were rinsed 15 times with phosphate buffered saline to remove residual test substance. Subsequently the skin tissues were incubated for 42 ± 2 h at 37°C.

Cytotoxic (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Positive (5% SDS), negative (ultrapure water) and quality (1% Triton X-100) control gave the appropriate response.

The relative mean tissue viability obtained after 60 minutes treatment with MDIPA Esterquat C18 unsatd. compared to the negative control tissues was 82.7 ± 1.5%. Since the mean relative tissue viability for the test substance was above 50%, MDIPA Esterquat C18 unsatd. is considered to be not irritating.