Amides, tall-oil fatty, N,N-di-Me

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-18 February 2013

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:

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

Version / remarks:

adopted 22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Amides, tall-oil fatty, N,N-di-Me

EC Number:

269-665-4

EC Name:

Amides, tall-oil fatty, N,N-di-Me

Cas Number:

68308-74-7

Molecular formula:

not applicable for UVCB

IUPAC Name:

Amides, tall-oil fatty, N,N-di-Me

Constituent 2

Reference substance name:

DMATO

IUPAC Name:

DMATO

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): DMATO
- Physical state: clear, brown liquid
- Analytical purity: 100%
- Lot/batch No.: 12J46399
- Expiration date of the lot/batch: October 2015
- Storage condition of test material: At room temperature (20±5°C) in the dark

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Buckman Laboratories, Lot No. 12J46399
- Expiration date of the lot/batch: 01/10/2015
- Purity test date: 100%; certificate of analysis dated 05/11/2012

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: assumed stable for study duration

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult-human derived epidermal keratinocytes

Details on animal used as source of test system:

Not applicable - in vitro study

Justification for test system used:

RhE model specified in the OECD Guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN RHE Model (skinEthic Laboratories, Lyon, France)
- Delivery date: 12/02/2013
- Date of initiation of testing: 12/02/2013

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of exposure each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco's phosphate buffered saline (DPBS). Rinsing was achieved by filling and emptying each tissue inert for approx. 40 seconds using a constant soft stream of DPBS to gently remove residual test material.
- Observable damage in the tissue due to washing: None.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT solution
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: none

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: water-killed tissues
- Procedure used to prepare the killed tissues (if applicable): untreated EPISKIN tissues were placed in a 12-well plate containing 2.2 mL of sterile distilled water, then incubated at 37°C, 5% CO2 in air for 48±1 hours.
- Result : Negligible intereference due to reduction of MTT by the test material occurred therefore the results were not used for quantitative correction of results

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability after 15 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritating to skin if the viability after 15 minutes exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

The test material was used as supplied, 10 µL was applied to the surface of the epidermis.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Tissues treated in triplicate

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

114.4

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean viability of the treated tissues was 114.4% after a 15 minute exposure period. The relative mean tissue viability for the positive control was 10.2% relative to the negative control and the standard deviation was 2.3%. The mean OD540 for the negative control was 0.834 and the standard deviation of the percent viability was 10.8%. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 8.5%. The acceptance criteria were therefore met for this assay.

Any other information on results incl. tables

An assessment found the test material was able to directly reduce MTT. An additional procedure using water-killed tissues was performed during the determination of skin corrosivity potential. The results obtained showed negligible interference due to direct MTT reduction. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

Table 1. Mean OD540 values and percentage viabilities

Item	OD540 of tissues	Mean OD540 of triplicate tissues	± SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control	0.752	0.834	0.090	90.2	100*	10.8
	0.819			98.2
	0.930			111.5
Positive Control	0.106	0.085	0.019	12.7	10.2	2.3
	0.080			9.6
	0.068			8.2
Test material	0.912	0.954	0.071	109.4	114.4	8.5
	0.913			109.5
	1.036			124.2

SD = standard deviation; * = mean viability of the negative control is set at 100%

Applicant's summary and conclusion

Interpretation of results:

other: non irritating

Conclusions:

The test material was considered to be non-irritating as the relative mean tissue viability was > 50% (114.4%).

Executive summary:

The skin irritating potential of DMATO was evaluated in the EPISKIN reconstructed human epidermis model, according to OECD Guideline 439 and GLP. Cell viability following exposure is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (within the mitochondria of viable cells) in treated tissues compared to relative controls.

10 µL of the test material was applied undiluted to the tissues for a 15 minute exposure period, followed by a 42 hour post-exposure incubation period. The test material, positive and negative controls were tested in triplicate. At the end of the post-exposure incubation period each tissue was taken for MTT loading. After MTT loading a total biopsy of each epidermis was made and placed into tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. At the end of the formazan extraction period each tubed was mixed and duplicate 200 µL samples were transferred to a 96 -well plate. The optical density was measured at 540 nm. The percentage viability is calculated from the MTT reduction in treated tissues relative to the negative control.

The relative mean viability of the treated tissues was 114.4%. The relative mean tissue viability was greater than 50%, therefore the test material is considered to be non-irritating under the conditions of the study.