N,N-bis(2-ethylhexyl)formamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Mar 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2019

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(2-ethylhexyl)formamide

EC Number:

860-695-9

Cas Number:

88247-41-0

Molecular formula:

C17H35NO

IUPAC Name:

N,N-bis(2-ethylhexyl)formamide

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek Corporation, Ashland, Massachusetts, USA

Source strain:

other: EpiDerm™ Skin Model (EPI-200)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200) (MatTek Corporation, Ashland, MA, USA)
- Tissue batch number: 34727
- Delivery date: 16 Mar 2021
- Date of initiation of testing: 17 Mar 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (3-min exposure) and 37 °C (60 +/-1 min exposure)
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: after the exposure, each tissue insert was rinsed twenty times with phosphate buffered saline (PBS)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 180 ± 5 min at 37 °C
- Spectrophotometer: Multimode Microplate Reader (FLUOstar Omega, BMG Labtech)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA (performed by the supplier MatTek Corporation, Ashland, Massachusetts, USA)
- Viability: The quality of the EpiDerm™ tissue was assessed by an MTT cell viability test (4 h, n = 3). The determined OD (540 - 570 nm) was 1.573 ± 0.084 (acceptance criteria 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.68 h (acceptance criteria: 3.68 – 8.02 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for biological contamination (bacteria, yeast and other funghi). No contamination was detected.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable as the test item did not show colour changing or reducing capacity after 60 min MTT incubation.

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

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered to be corrosive to skin if the viability after 3-minutes exposure is less than 50% and the viability after 1-hour exposure is less than 15%.
- The test item is considered to be non-corrosive to skin if the viability after 3-minutes exposure is greater than or equal to 50% and the viability after 1-hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8.0 N

Duration of treatment / exposure:

3 min and 60 ± 1 min

Number of replicates:

Duplicates for each treatment and control group

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

3 min exposure

Value:

104.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

60 min exposure

Value:

91.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The test item did not show reducing capacity after 1 h incubation with MTT
- Colour interference with MTT: The test substance did not change colour, when mixed with deionised water.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The laboratory has demonstrated technical proficiency in the EpiDerm skin corrosion test by correct prediction of the corrosive potential of proficiency chemicals and by correct assignment of the chemicals into the sub-classes of corrosiveness as defined by the OECD guideline 431.

ACCEPTANCE OF RESULTS: (refer to Table 1 under "Any other information on results incl. tables")
- Acceptance criteria met for negative control: The mean OD 570 nm of the tissue replicates treated with the negative control was 1.766 for the 3 min exposure and 1.937 for the 1 h exposure and fell within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was < 15% compared to the negative control (1.6% for 1 hour exposure).
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation (CV) in the range 20 – 100% viability between tissue replicates was ≤ 30% (values between 1.0 – 28.9%).

Any other information on results incl. tables

Table 1: Results of the skin corrosion test

Group	Tissue No.	3 min exposure						60 min exposure
		OD			Cell viability (%)			OD			Cell viability (%)
		individual	mean		mean ± SD		CV	individual	mean		mean		CV
Distilled water	1	1.64	1.687	1.766	95.5	100.0 ± 6.29	6.3	1.952	1.95	1.937	100.7	100.0 ± 0.99	1.00
		1.662						1.948
		1.76						1.951
	2	1.848	1.844		104.4			1.904	1.923		99.3
		1.899						1.898
		1.785						1.967
8 N KOH	1	0.101	0.099	0.125	5.6	7.1 ± 2.05	28.9	0.032	0.032	0.030	1.7	1.6 ± 0.21	13.3
		0.099						0.032
		0.098						0.031
	2	0.149	0.150		8.5			0.029	0.028		1.4
		0.154						0.028
		0.148						0.028
Test item	1	1.896	1.883	1.839	106.6	104.1 ± 3.54	3.4	1.755	1.746	1.769	90.1	91.3 ± 1.70	1.9
		1.878						1.738
		1.876						1.746
	2	1.771	1.795		101.6			1.804	1.792		92.5
		1.809						1.795
		1.805						1.777
CV: coefficient of variation; OD: optical density; SD: standard deviation

 

Table 2: Historical control data generated in the testing facility from May 2018 - Jan 2021 (n = 20)

	OD
	3-minute	exposure	60-minute exposure
	Negative control	Positive control	Negative control	Positive control
Mean	1.908	0.134	1.887	0.051
SD	0.145	0.039	0.149	0.017

OD: optical density; SD: standard deviation

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat. 1, 1A, 1B/C) based on a positive result in the Reconstructed human epidermis test method (in vitro skin corrosion). A negative in vitro corrosivity response is not conclusive with respect to non-classification or classification as a skin irritant and therefore requires further evaluation and/or data generation.