2-(N-ethylanilino)ethanol

Endpoint summary

Description of key information

The test item showed irritant and corrosive effects in two separate in-vitro EpiDerm studies. The eye irritancy potential of 2-(N-ethylanilino)ethanol was investigated in the bovine corneal opacity and permeability assay. All 3 corneas treated with 2-(N-ethylanilino)ethanol showed moderate opacity of the tissue and white stripes on the cornea were visible.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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)

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

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

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.

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%

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%

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%

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

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

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.

Reference 2

Endpoint:

skin irritation: 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

28 July, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

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 No.: 28637).

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): 47 µL/cm2

NEGATIVE CONTROL
- Dulbecco’s phosphate buffered saline (DPBS; Gibco, Cat. No. 14040-091, Lot No.: 1838067).

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 5% sodium dodecyl sulfate (TC-SDS-5%; MatTek, CAS No.: 151-21-3, Lot No.: 040418SVA).

Duration of treatment / exposure:

60 ± 1 minutes

Duration of post-treatment incubation (if applicable):

The plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

2.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

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

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item showed irritant effects.

Executive summary:

In the present study 2-(N-ethylanilino)ethanol was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.  

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%.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (2.3%) after 60 min treatment and 42 h post-incubation.  

The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.911). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (2.7%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.1% – 1.9%).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the first skin corrosion 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 skin corrosion 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.

Although effects were seen in the Bovine Corneal Opacity and Permeability Assay, no prediction could be made regarding the classification of 2-(N-ethylanilino)ethanol for eye irritation according to the evaluation criteria. Mean in-vitro irritation score was 7.95, which is > 3, but much lower than the upper limit for category 1 irritation of 55. Hence, this result contradicts the outcome of corrosive for skin based on the EpiDerm tests.

Justification for classification or non-classification

In the in-vitro skin corrosion test, the mean relative tissue viability (% negative control) was reduced below 15% after 60 min treatment but remained above 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. Although no conclusive prediction could be made regarding the classification of 2-(N-ethylanilino)ethanol for eye corrosion, the results of the skin corrosion tests resulted in classification as eye corrosive.