Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2016 to 08 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 431: In Vitro Skin Corrosion: reconstructed human epidermis (RHE) test method (adopted 28 July 2015).

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:

European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

EC Number:

701-003-6

Cas Number:

1454803-04-3

Molecular formula:

C20H39NO3 to C26H51NO3

IUPAC Name:

Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

Test material form:

liquid

Details on test material:

Identification: MLA-3202
Appearance: Clear amber-red liquid
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until 17 February 2019 (expiry date)
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)
CAS Number: 1454803-04-3
Test item handling No specific handling conditions required

Specific details on test material used for the study:

Batch: RC-1045Study specific test item informationPurity/composition correction factor: No correction factor requiredChemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)CAS Number: 1454803-04-3Test item handling: No specific handling conditions required

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

other: Not applicable

Details on animal used as source of test system:

Test system EpiDerm Skin Model (EPI-200, Lot no.: 23697 kit U and T). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered 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. Source MatTek Corporation, Ashland MA, U.S.A.

Justification for test system used:

Rationale Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TissuesOn 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. 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, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 69 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.2 - 36.4°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.

Control samples:

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

50 μl

Duration of treatment / exposure:

Two tissues were used for a 3-minute exposure to MLA-3202 and two for a 1-hour exposure.

Duration of post-treatment incubation (if applicable):

Tissues were incubated for 3 hours at 37°C in air containing 5% CO2.

Number of replicates:

2 per application time/group

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour treatment

Value:

86

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute treatment

Value:

67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MLA-3202 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that MLA-3202 did not interfere with the MTT endpoint. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with MLA-3202 compared to the negative control tissues was 67% and 86% respectively. Because the mean relative tissue viability for MLA-3202 was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment MLA-3202 is considered to be not corrosive. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 7%. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 5%, indicating that the test system functioned properly.

Any other information on results incl. tables

Mean absorption in thein vitroskin corrosion test with MLA-3202

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	2.144	2.239	2.192	±	0.067	2.033	2.081	2.057	±	0.034
MLA-3202	1.439	1.496	1.467	±	0.040	1.764	1.756	1.760	±	0.006
Positive control	0.146	0.152	0.149	±	0.004	0.141	0.218	0.179	±	0.055

SD = Standard deviation

Duplicate exposure are indicated by A and B.

In this table the values are corrected for background absorption (0.0405). Isopropanol was used to measure the background absorption.

 

Mean tissue viability in thein vitroskin corrosion test with MLA-3202

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
MLA-3202	67	86
Positive control	7	9

 

Coefficient of Variation between tissue replicates

	3 minute	1 hour
Negative control	4.3	2.3
MLA-3202	3.8	0.5
Positive control	3.9	35.5

CV (%) = 100 – [(lowest OD570/highest OD570) x 100%]

 

INDIVIDUAL OD MEASUREMENTS AT 570 NM

	3-minute application (OD570)		1-hour application (OD570)
	A	B	A	B
Negative control OD570measurement 1 OD570measurement 2 OD570measurement 3	2.1973 2.1816 2.1752	2.3017 2.2773 2.2608	2.1016 2.0743 2.0458	2.1050 2.1339 2.1257
MLA-3202 OD570measurement 1 OD570measurement 2 OD570measurement 3	1.4956 1.4680 1.4756	1.5285 1.5558 1.5246	1.8073 1.7824 1.8248	1.8057 1.7708 1.8121
Positive control OD570measurement 1 OD570measurement 2 OD570measurement 3	0.1862 0.1874 0.1870	0.1939 0.1910 0.1934	0.1827 0.1816 0.1792	0.2583 0.2639 0.2537

OD – Optical density

Duplicate exposure are indicated by A and B.

 

HISTORICAL CONTROL DATA FOR IN VITRO SKIN CORROSION STUDIES

	Negative control		Positive control		Positive control
	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (% viability)	1-hour treatment (% viability)
Range	1.324 – 2.615	1.361 – 2.352	0.172 – 0.56	0.057 – 0.277	6 – 22	3 – 12
Mean	1.86	1.86	0.18	0.13	10.67	7.17
SD	0.24	0.22	0.10	0.05	3.9	2.36
n	65	67	64	61	30	30

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of December 2012 to December 2015.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Finally, it is concluded that this test is valid and that MLA-3202 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

This objective of the study was to evaluate the corrosivity potential of MLA-3202 using a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of MLA-3202 was tested through topical application for 3 minutes and 1 hour. This method was designed to be compatible with the following:

-OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method” (adopted 28 July 2015)

-Method B.40 of Commission Regulation (EC) No. 440/2008

 

Batch RC-1045 of MLA-3202 was a clear amber-red liquid. MLA-3202 was applied undiluted (50 μl) directly on top of the skin tissue.

 

The positive control had a mean relative tissue viability of 7% after 3 minutes of exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of 20 - 100% viability, the Coefficient of Variation between tissue replicates was ≤ 5%, indicating that the test system functioned properly.

 

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with MLA-3202 compared to the negative control tissues was 67% and 86%, respectively. Because the mean relative tissue viability for MLA-3202 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment MLA-3202 is considered to be not corrosive.

 

Finally, it is concluded that this test is valid and that MLA-3202 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.