1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C16-18 (even numbered) and C18 unsatd., 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

(20 July 2012)

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

Test material

Test material information

Constituent 1

Reference substance name:

1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C16-18 (even numbered) and C18 unsatd., Me sulfates (salts)

EC Number:

941-174-6

IUPAC Name:

1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C16-18 (even numbered) and C18 unsatd., Me sulfates (salts)

Test material form:

other: white solid wax

Details on test material:

- Name of test material: MDIPA-Esterquat C16-18 and C18 unsatd.
- Physical state: waxy solid
- Analytical purity: 100%

Specific details on test material used for the study:

- Name of test material: MDIPA-Esterquat C16-18 and C18 unsatd.
- Physical state: solid
- Analytical purity: 100%

Test animals

Species:

other: three-dimensional human epidermis model (EPISKIN-SM)

Test system

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Controls:

other: negative control, positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
To reach optimal contact with the tissue without causing mechanical damage, the test substance was applied using a cotton swab. The exact amount of test substance applied could not be determined using this method. Since optimal contact was obtained using this method this deviation has no impact on the study integrity.
- skin was moistened with 5 μL Milli-Q water to ensure close contact

Duration of treatment / exposure:

15 min

Observation period:

42 h

Number of animals:

triplicates

Details on study design:

TEST SYSTEM
- EPISKIN Small Model (EPISKIN-SM, 0.38 cm², Batch no.: 12-EKIN-039)
Three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

APPLICATION
-the test substance was heated to approximately 50°C to soften the waxy substance
- the test substance was applied with a cotton swab directly on top of the moistened skin tissue and spread to match the size of the tissue
- incubation with test substance for 15 min at room temperature
- after washing: post incubation period of a total of 42 h at 37°C
- cell viability was determined by using the standard MTT assay

Negative control (3 tissues): 25 µL PBS
Positive control (3 tissues): 25 µL 5% SDS

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the tissues were washed with phosphate buffered saline, pre-warmed to approximately 50°C, removal was performed with a spatula (this has no impact on the study integrity)
- Time after start of exposure: 15 min

MTT ASSAY
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-medium (0.3 mg/mL). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm.

DATA ANALYSIS
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

SCORING SYSTEM:
- mean tissue vialbility > 50% = non-irritant
- mean tissue viability

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Value:

114

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Value:

9

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 114%. Since the mean relative tissue viability was above 50%, the test item is considered to be non-irritant.
The positive control had a mean cell viability after 15 minutes exposure of 9%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 14%, indicating that the test system functioned properly.

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

Any other information on results incl. tables

Viability of epidermal models:

	Mean tissue viability (% of control)
Negative control (PBS)	100
Positive control (5% SDS)	9
Test item	114

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

MDIPA-Esterquat C16-18 and 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 performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (22 July 2010) and EU method B.46 (In vitro skin irritation: reconstructed human epidermis model test) (20 July 2012), MDIPA-Esterquat C16-18 and C18 unsatd. (100% a. i.) was applied to the three-dimensional human epidermis model tissue (EPISKIN Small Model (EPISKIN-SM, 0.38 cm², Batch no.: 12-EKIN-039) for an exposure period of 15 minutes. The number of replicate tissues was three). The test substance was heated in advance to approximately 50°C to soften the waxy substance. To achieve optimal contact with the tissue without causing mechanical damage, the test substance was applied using a cotton swab. The exact amount of test substance applied to cover the entire skin surface could not be determined using this method. Since optimal contact was obtained using this method this deviation has no impact on the study integrity. After 15 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline pre-warmed to approximately 50°C, and residual test substance was carefully removed using a spatula. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (PBS) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment with MDIPA-Esterquat C16-18 and C18 unsatd. compared to the negative control tissues was 114%. Since the mean relative tissue viability for the test substance was above 50%, MDIPA-Esterquat C16-18 and C18 unsatd. is identified to be not irritating.

Data generated by fully validatedin vitromethods can be used for REACH purposes provided that the information for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment. Based on the result of thein vitroskin irritation study, MDIPA-Esterquat C16-18 and C18 unsatd. as neat substance would be Not Classified (NC) for skin irritation.