2-(N-ethylanilino)ethanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

29 July, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-(N-ethylanilino)ethanol

EC Number:

202-160-9

EC Name:

2-(N-ethylanilino)ethanol

Cas Number:

92-50-2

Molecular formula:

C10H15NO

IUPAC Name:

2-(N-Ethylanilino)ethanol

Test material form:

liquid - solid: mixture of

Details on test material:

At room temperature, the substance can be either a primrose yellow solid or a liquid.

Specific details on test material used for the study:

Batch No.: 18052904
Physical State at RT: liquid, the test item can be either solid or liquid. At RT, the test material was a viscous liquid that could be pipetted. Thus, the test material was tested as a liquid and applied undiluted.
Colour: primrose yellow
Storage Conditions: room temperature, protected from light
Retest Date: 28 May 2019

In vitro test system

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Cell source:

other: Sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm2); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot: 28646 for exp. 1, 28651 for exp. 2).

Justification for test system used:

The EpiDerm epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.

Vehicle:

unchanged (no vehicle)

Details on test system:

This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL dispensed directly atop the EpiDerm tissue. The test item was spread to match size of the tissue.
NEGATIVE CONTROL
- distilled water
POSITIVE CONTROL
- 8 N Potassium Hydroxide (KOH; CAS No.: 1310-58-3; Lot: 10357-004, NeoLab)

Duration of treatment / exposure:

3 min and 60 min exposure time

Number of replicates:

The test was performed on a total of 4 tissues per dose group, 2 replicates for each treatment period

Test system

Details on study design:

3 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 sec. was kept between dosing. After 3 min of application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 µL pre-warmed assay medium per well. All inserts were treated in the same manner. Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 µL pre-warmed MTT solution. The plate was incubated for 3 h at 37± 1°C, 5.0% CO2 / 95% air.
60 min experiment: after 60 min application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 µL pre-warmed assay medium per well. All inserts were treated in the same manner. Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 µL pre-warmed MTT solution. The plate was incubated for 3 h at 37 ± 1°C, 5.0% CO2 / 95% air.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 - 3 min Experiment

Value:

78.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

9.8%

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 - 60 min Experiment

Value:

15.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

6.3%

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2 - 3 min Experiment

Value:

104.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

7.3%

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2 - 60 min Experiment

Value:

7.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

2.3%

Other effects / acceptance of results:

Mean Absolute OD570 nm NK (3 min Experiment): 1.680 (Experiment 1); 1.573 (Experiment 2) / 0.8 ≤ NK ≤ 2.8 => pass
Mean Absolute OD570 nm NK (60 min Experiment): 1.723 (Experiment 1); 1.665 (Experiment 2) / 0.8 ≤ NK ≤ 2.8 => pass
Mean Relative Tissue Viability [%] of PC (60 min experiment): 6.3 (Experiment 1); 2.3 (Experiment 2)/ < 15% => pass
CV [%] (in the range of 20 – 100% viability): 3.1 – 5.0 (Experiment 1); 1.3 – 1.5 (Experiment 2)/ ≤ 30% => pass

Any other information on results incl. tables

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. The mixture of 25 mg of the test item per 300 µl aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

Table 1: Results of the main experiment:

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.855	1.901	1.951	0.095	0.094	0.092	0.083	0.089	0.090
	1.903	1.914	1.945	0.099	0.096	0.096	0.085	0.090	0.090
OD570(Blank Corrected)	1.811	1.857	1.907	0.051	0.050	0.048	0.039	0.045	0.046
	1.859	1.870	1.901	0.055	0.052	0.052	0.041	0.046	0.046
Mean OD570of the Duplicates (Blank Corrected)	1.835	1.863	1.904	0.053	0.051	0.050	0.040	0.045	0.046
Total Mean OD570of 3 Replicate Tissues (Blank Corrected)	1.867*			0.051			0.044
SD OD570	0.035			0.002			0.004
Relative Tissue Viability [%]	98.3	99.8	102.0	2.8	2.7	2.7	2.1	2.4	2.5
Mean Relative Tissue Viability [%]	100.0			2.7**			2.3
SD Tissue Viability [%]***	1.9			0.1			0.2
CV [% Viabilities]	1.9			3.2			8.1

*  Blank-corrected mean OD570 nmof the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is£ 20%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is≤ 18%

Table 2: Quality criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nmNK	1.911	0.8 ≤ NK ≤2.8	pass
Mean Relative Viability [%] PC	2.7	≤ 20%	pass
SD Viability[%]	0.1 – 1.9	≤ 18%	pass

Applicant's summary and conclusion

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 15% after 60 min treatment but not below 50% after 3 min treatment. The test item is therefore classified as “corrosive“ in accordance with a combination of optional UN GHS sub-categories 1B and 1C.

Executive summary:

In the present study the skin corrosivity potential of 2-(N-ethylanilino)ethanol was analysed. Since corrosive chemicals are cytotoxic after a short time exposure to the stratum corneum of the epidermis the cytotoxic effects of the test item on EpiDermÔ, a reconstituted three-dimensional human epidermis model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 min and 60 min exposure period and compared to those of the concurrent negative controls.

The mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple.Therefore, NSMTT equaled 0%.

The mixture of 50 µL test item per 300 µL Aqua dest.and per 90 µL isopropanolshowed no colouring as compared to the solvent.Therefore NSC equaled 0%.

In the first experiment, the test item showed no corrosive effects. The mean relative tissue viability (% negative control) was³ 50% (78.7%) after 3 min treatment and³ 15% (15.5%) after 60 min treatment. However, the mean viability (15.5%) after 60 min treatment was near to the threshold of 15% and also the two tissue replicates showed inconclusive results. One tissue replicate was above (15.9%) and one below (14.4%) the 15% threshold after 60 min treatment. Thus, as recommended by the OECD guideline, a second experiment was performed.

In the second experiment, the test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 15% (7.4%) after 60 min treatment but not below 50% (104.9%) after 3 min treatment. Both tissue replicates were below (7.3% and 7.5%) the threshold of 15% after 60 min treatment.