Amides, C8-18 (even-numbered), C18unsatd, N-[3-(dimethylamino)propyl]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-11-24 to 2005-11-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline no. 431: In Vitro Skin Corrosion: Human Skin Model Test (adopted 13 April 2004)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

(8 June 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Amides, C8-18 even numbered, N-[3-(dimethylamino)propyl]

EC Number:

930-947-3

IUPAC Name:

Amides, C8-18 even numbered, N-[3-(dimethylamino)propyl]

Test material form:

other: pasty solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: cultered cells

Details on test system:

In vitro Test system EpiDerm Skin Model (EPI-200, Lot no.: 6454, kit C, source MatTek Corporation, Ashland MA, USA) used.

The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-Iayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Cell culture:
- Tissues:
-- On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM medium.

- Media:
-- DMEM (Dulbecco's Modified Eagle's Medium) = Supplemented DMEM medium, serum-free supplied by MatTek Corporation.
-- MTT medium = MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation..

- Environmental conditions:
-- All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 78 - 89%), containing 5.0 ±0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.6 - 37.0°C). Temporary deviations from the humidity (with a maximum of 20%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

the test item was heated to 37°C and applied undiluted (50 µL) directly on top of the skin tissue.

Duration of treatment / exposure:

The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour.

Details on study design:

Study design
The skin tissues were kept in the refrigerator the day they were received. The next day, at least one hour before the assay is started the tissues were transferred to 6-well plates containing 0.9 mL DMEM medium per well. The level of the DMEM medium is just beneath the tissue. The medium was replaced with fresh DMEM medium just before test item was applied. The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a three minutes exposure to test item and two for a one hour exposure. 50 µL of the undiluted test substance (heated to 37°C) was added (with a pipette) into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 µl Milli-Q water (negative control) and with 50 µL 8N KOH (positive control), respectively. After the exposure period, the tissues were washed with phosphate buffered saline (PBS, Gibco) to remove residual test substance. Rinsed tissues were kept in 24 well plates on 300 µL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

Cell viability measurement
The DMEM medium was replaced by 300 µL MTT-medium and tissues were incubated for 3 h at 37°C in air containing 5 ± 0.5% carbon dioxide. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 540 nm in triplicate with the Multiskan Spectrum (Thermo Labsystems). Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

Interpretation
- Acceptability of the assay: The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD540 of the two tissues of the negative control should be OD >/= 0.8.
b) The mean relative tissue viability of the 3 minutes exposure of the positive control should be c) The maximum inter tissue variability (in viability) is 30% between two tissues treated identically.
d) The maximum difference in percentage between the mean viability of two tissues and one of the two tissues is 15%..

- Data evaluation and statistical procedures:
-- A test substance is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3 min treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test substance considered non-corrosive after 3 min (viability >/= 50%) treatment is considered corrosive if the mean relative tissue viability after 1 hr treatment with the test substance is decreased below 15%.
-- A test substance is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3 min treatment compared to the negative control tissues is not decreased below 50%.,
b) In addition, the mean relative tissue viability after 1 hr treatment is not decreased below 15%

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test substance; exposure time: 3 minutes

Value:

45

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

not specified

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test substance; exposure time: 1 hour

Value:

45

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

not specified

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative control (water): exposure time: 1 hour

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

not specified

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control (potassium hydroxide); exposure time: 3 minutes

Value:

7

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

not specified

Any other information on results incl. tables

Mean absorption in the in vitro skin corrosion test with test item

	3 min application				1 hour application
	A	B	mean	SD	A	B	mean	SD
Negative control	1.476	1.487	1.481	±0.008	1.446	1.387	1.417	±0.042
Test substance	0.687	0.658	0.673	±0.020	0.655	0.624	0.639	±0.022
Positive control	0.097	0.099	0.098	±0.001	0.089	0.090	0.090	±0.001

 

SD = Standard deviation

 

In this table the values are corrected for background absorption. Isopropanol was used to measure the background absorption.

 

Mean tissue viability in the in vitro skin corrosion test with test item

	3 min application viability (percentage of control)	1 hour application viability (percentage of control)
Negative control	100	100
Test substance	45	45
Positive control	7	6

 

This table shows the mean tissue viability obtained after 3 minutes and 1 hour treatment with test item compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3 minutes and 1 hour treatment with the test item compared to the negative control tissues was 45% for both treatment times. Since the mean relative tissue viability for the test item was below 50% after 3 minutes treatment it is considered to be corrosive.

The absolute mean OD540(Optical density) of the negative control was higher than 0.8. The mean relative tissue viability of the 3 minutes exposure of the positive control was less than 30%. The maximum inter tissue variability in viability between two tissues treated identically was less than 30% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 15%. It was therefore concluded that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

corrosive

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

It is concluded that this test is valid and that test substance Amides, C8-18, N-[3-(dimethylamino)propyl] is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

In the In Vitro Skin Corrosion: Human Skin Model Test according to OECD Guideline 431 (adopted 13 April 2004) and EU Method B.40: "Skin corrosion" (8 June 2000), the topical application of Amides, C8-18, N-[3-(dimethylamino)propyl] on the skin tissue for a 3 minutes and 1 hour exposure was performed, followed by immediate determination of the cytotoxic (corrosive) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3 minutes and 1 hour treatment with the test item compared to the negative control tissues was both 45%. Since the mean relative tissue viability for the test item was below 50% after 3 minutes treatment with the test item is considered to be corrosive.

The positive control had mean relative tissue viability after 3 minutes exposure of less than 30%. The negative control had a mean OD540 (optical density at 540 nm) higher than 0.8. The maximum inter tissue variability in viability between two tissues treated identically was less than 30% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 15%, indicating that the test system functioned properly.

It is concluded that this test is valid and that the test substance is corrosive in the in vitro skin corrosion test under the experimental conditions described.