C8-18 (even numbered) and C18 (unsaturated) sarcosinates, 2-hydroxypropylammonium salt

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:

other: GLP guideline study without restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-(N-methyldodecanamido)acetic acid 2-hydroxypropan-1-aminium 2-(N-methyldodecanamido)acetate

EC Number:

943-406-1

Molecular formula:

Not applicable. This substance is UVCB.

IUPAC Name:

2-(N-methyldodecanamido)acetic acid 2-hydroxypropan-1-aminium 2-(N-methyldodecanamido)acetate

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): MIPA-Cocoyl-Sarkosinat
- Substance type: organic (UVCB)
- Physical state: clear yellow-brown viscous liquid
- Analytical purity: 100%, according to definition of UVCB
- Impurities (identity and concentrations): no impurities, UVCB
- Lot/batch No.: 070715
- Expiration date of the lot/batch: 31 Dec 2016
- Stability under test conditions: stable in vehicle water
- Storage condition of test material: at room temperature
- Other: pH 5.7 - 6.0 (1% in water)

Test animals

Species:

human

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

TEST SYSTEM
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm², Batch no.: 16-EKIN-005 (first test), 16-EKIN-008 (second test) and 16-EKIN-010 (third test).
This model is a 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.

SOURCE
SkinEthic Laboratories, Lyon, France

PREPARATION AND PREINCUBATION
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 27, 22 and 23 hours (first, second and third test, respectively) at 37 °C. Maintenance medium and Assay medium were supplied by SkinEthic Laboratories, Lyon, France.

ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test item exposure for 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 60 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.4 - 37.2 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Type of coverage:

other: not applicable, in vitro human skin model

Preparation of test site:

other: not applicable, in vitro human skin model

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable, in vitro test system. Negative control tissues treated with phosphate buffered saline (PBS)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL
- Concentration (if solution): undiluted test item (100%)

Duration of treatment / exposure:

15 ± 0.5 minutes

Observation period:

Not applicable, in vitro test system

Number of animals:

3 replicate tissues per test item

Details on study design:

APPLICATION/TREATMENT OF THE TEST ITEM
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty-five µL of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µL PBS (negative control) and 3 tissues with 25 µL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with PBS to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37 °C.

CELL VIABILITY MEASUREMENT
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-solution (0.3 mg/mL in PBS). 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 69 – 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

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

ACCEPTABILITY OF THE ASSAY
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤ 18.
b) The mean relative tissue viability of the positive control should be ≤ 50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤ 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤ 18.

DATA EVALUATION
A test item is considered at least irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
In that case no decision on a classification into Category 1 or Category 2 can be made; additional information on corrosion is needed.

A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: cell viability

Value:

59

Remarks on result:

other:

Remarks:

Basis: mean 3 tissue replicates. Time point: 15 ± 0.5 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: 1st test. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: cell viability

Value:

21

Remarks on result:

other:

Remarks:

Basis: mean 3 tissue replicates. Time point: 15 ± 0.5 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: 1st test positive control. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: cell viability

Value:

100

Remarks on result:

other:

Remarks:

Basis: mean 3 tissue replicates. Time point: 15 ± 0.5 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: 1st test negative control. (migrated information)

Results 4

Irritation / corrosion parameter:

other: other: cell viability

Value:

11

Remarks on result:

other:

Remarks:

Basis: mean 3 tissue replicates. Time point: 15 ± 0.5 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: 2nd test. (migrated information)

Results 5

Irritation / corrosion parameter:

other: other: cell viability

Value:

17

Remarks on result:

other:

Remarks:

Basis: mean 3 tissue replicates. Time point: 15 ± 0.5 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: 2nd test positive control. (migrated information)

Results 6

Irritation / corrosion parameter:

other: other: cell viability

Value:

100

Remarks on result:

other:

Remarks:

Basis: mean 3 tissue replicates. Time point: 15 ± 0.5 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: 2nd test negative control. (migrated information)

Results 7

Irritation / corrosion parameter:

other: other: cell viability

Value:

10

Remarks on result:

other:

Remarks:

Basis: mean 3 tissue replicates. Time point: 15 ± 0.5 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: 3rd test. (migrated information)

Results 8

Irritation / corrosion parameter:

other: other: cell viability

Value:

19

Remarks on result:

other:

Remarks:

Basis: mean 3 tissue replicates. Time point: 15 ± 0.5 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: 3rd test positive control. (migrated information)

Results 9

Irritation / corrosion parameter:

other: other: cell viability

Value:

100

Remarks on result:

other:

Remarks:

Basis: mean 3 tissue replicates. Time point: 15 ± 0.5 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: 3rd test negative control. (migrated information)

In vivo

Irritant / corrosive response data:

First test: Mean tissue viability of 59% after treatment with the test item. Since two of the individual viabilities were above 50% and one was below 50%, no conclusion could be made and a repeat test was performed.
Second test: Mean tissue viability of 11% after treatment with the test item. Since the difference with the first test was 48% and the test item was viscous a repeat test with additional washing was performed, to be sure that the test item was removed properly.
Third test: Mean tissue viability of 10% after treatment with the test item. The third test confirmed the results obtained in the second test.
The Standard deviations in all treatment groups in all tests were well below 18% (maximum < 11%), indicating that the test system functioned properly.

Since once equivocal results and two times clear positive results were obtained, it is concluded that these tests were valid and that the test item is at least irritant in the in vitro skin irritation test under the experimental conditions described in this report.

However, based on the results of this study no conclusion can be made regarding a classification into Category 1 or Category 2; therefore, further information on skin corrosion is required.

Any other information on results incl. tables

Table: Mean tissue viability in the in vitro skin irritation test

	Mean tissue viability (percent of control)	Standard deviation
First test
Negative control	100	3.6
Test item	59	10.4
Positive control	21	3.5
Second test
Negative control	100	7.0
Test item	11	1.2
Positive control	17	1.3
Third test
Negative control	100	8.1
Test item	10	4.4
Positive control	19	6.9

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU