2-(2-(dimethylamino)ethoxy)-N-(2-(2-(dimethylamino)ethoxy)ethyl)-N-methylethanamine

Endpoint summary

Description of key information

Skin irritation/corrosion: The substance was observed to be corrosive in the in vitro EPIDERM^TM Skin Corrosion Test performed according to OECD Guideline 431 and EU Method B.40bis (Warren, 2017).

Eye irritation: The study does not need to be conducted because the substance is classified as skin corrosion, leading to classification as serious eye damage (category 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-09 to 2017-08-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

yes

Remarks:

inter-tissue variability in viability for 3-minutes exposure to test item > 30% (i.e. 31.8%), however no influence on classification (relative mean viability >50% for this group)

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS:"In vitro Skin Corrosion: Human Skin Model Test"

Version / remarks:

30 May 2008

Deviations:

yes

Remarks:

inter-tissue variability in viability for 3-minutes exposure to test item > 30% (i.e. 31.8%), however no influence on classification (relative mean viability >50% for this group)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- CAS number: 65286-55-7
- Physical state/appearance: clear colorless liquid
- Source and lot/batch No.of test material: DR74271215
- Expiration date of the lot/batch: 2018-12-30
- Purity: 98.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was used as supplied.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

Cell type: epithelial, derived from human skin, and formed into a stratified, cornified epithelium

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: MatTek
- Tissue batch number(s): 25835
- Production date: no data
- Delivery date: 2017-08-08 (Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator (2 to 10 °C) until use)
- Date of initiation of testing: 2017-08-09
- Assay Medium lot number: 080317TMA

TEMPERATURE USED FOR TEST SYSTEM
- Pre-incubation: The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-Minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the
tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- MTT loading: The plate was incubated (37 °C, 5% CO2) for 3 hours.
After the 3-Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.
- Absorbance/optical density measurements:
After extraction, each tissue was pierced with a pipette fitted with a 1000 μL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 μL aliquots of the extract were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader.

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 of test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): for both 60-Minute and 3-Minute exposure period: 50 μL of sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): for both 60-Minute and 3-Minute exposure period: 50 μL of 8.0 N Potassium Hydroxide

Duration of treatment / exposure:

3 minutes and one hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

duplicates: 4 tissues per test item, negative control and positive control: 2 for the 3-minute exposure period and 2 for the 1 hour-exposure

Details on study design:

STUDY DESIGN
Pre-test procedure

Assessment of Direct Test Item Reduction of MTT :
MTT Dye Metabolism, Cell Viability Assay The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction :
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below: 50 µL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was t ested concurrently to act as a control. If the MTT solution containing the test item turns blue/purple relative to the control, the test item was presumed to have reduced the MTT. The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and freeze- killed tissues.

This step was a functional check which employs freeze-killed tissues that possess no metabolic act ivity but absorb and bind the test item like viable tissues. Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12 well plate and storing in a freezer (14 to 30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature. In addition to the normal test procedure, the MTT reducing test item was applied to two freeze killed tissues per exposure period. In addition, two freeze killed tissues per exposure period remained untreated. The untreated freeze killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

Assessment of Color Interference with the MTT Endpoint :
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 50 µL of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 oC, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

Main Test
Pre-Incubation
The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3 Minute and 60 Minute exposure periods. EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of Test Item and Rinsing
Before pre-incubation was complete, a 24 well plate was prepared for use as a “holding plate” for both the 3 Minute and 60 Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24 well plate was prepared for the MTT loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required. After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 Minute exposure period was returned to the incubator, while the other was being dosed for the 60 Minute exposure. For the 60 Minute exposure peri od, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure an d to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60 Minute exposure period. When dosing for the 60 Minute exposure period was complete, the same procedure was repeated for the 3 Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24 well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60 Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated. After the 3 Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

Absorbance/Optical Density Measurements
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application

Value:

75.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application

Value:

12.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: coefficient of variation

Run / experiment:

3-minute application

Value:

31.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Irritation / corrosion parameter:

other: Coefficient of Variation

Run / experiment:

1-hour application

Value:

5.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Irritation / corrosion parameter:

other: optical density

Run / experiment:

3-minute application

Value:

1.078

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: optical density

Run / experiment:

1-hour application

Value:

0.193

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

3-minute application:
- relative mean viability:
negative control: The mean % viability of the negative control tissue is set at 100%
positive control: 6.4
- coefficient of variation between tissue replicates
negative control: 15.2
positive control: not applicable
- mean optical density:
negative control: 1.423
positive control: 0.091

1-hour application:
- relative mean viability:
negative control: The mean % viability of the negative control tissue is set at 100%
positive control: 0.9
- coefficient of variation between tissue replicates
negative control: 3.7
positive control: not applicable
- mean optical density:
negative control: 1.567
positive control: 0.014

Results: An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes. The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference. The mean viability of the negative control tissues is set at 100%

- OTHER EFFECTS:
- Direct-MTT reduction:
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.
- Colour interference with MTT:
The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The mean OD570 for the negative control treated tissues was 1.423 for the 3-Minute exposure period and 1.567 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control:
The relative mean tissue viability for the positive control treated tissues was 0.9% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements:
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30% with the exception of the test item treated 3 minute exposure group which was 31.8% However, as the relative tissue viability of this group was considerably higher than 50% this failure would not affect the overall classification of corrosivity. This acceptance criterion was therefore not fully satisfied and is reported as a deviation (see above in text field Any other information on materials and methods incl. tables).

Direct MTT Reduction

An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze‑killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze‑killed tissues for quantitative correction of results or for reporting purposes.

Assessment of color interference with the MTT

The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

Test item, positive control item and negative control item

Mean OD562 values and viabilities for the negative control, positive control and test item are given below:

Tissue	Exposure Period	MeanOD570of individual tissues	Mean OD570of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.575	1.423	0.216	15.2	100*
		1.270
	60 Minutes	1.526	1.567	0.057	3.7
		1.607
Positive Control	3 Minutes	0.076	0.091	0.021	na	6.4
		0.105
	60 Minutes	0.013	0.014	0.001	na	0.9
		0.015
Test Item	3 Minutes	1.320	1.078	0.343	31.8	75.7
		0.835
	60 Minutes	0.186	0.193	0.010	5.1	12.3
		0.200

OD: Optical density

*: The mean percentage viability of the negative control tissue is set at 100%

na: Not applicable

The relative mean viabilities of the test item treated tissues were as follows:

 3 minutes exposure: 75.7%

60 minutes exposure: 12.3%

     

Quality Criteria

The mean OD570 for the negative control treated tissues was 1.423 for the 3‑Minute exposure period and 1.567 for the 60‑Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 0.9% relative to the negative control following the 60‑Minute exposure period. The positive control acceptance criterion was therefore satisfied.

In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30% with the exception of the test item treated 3 minute exposure group which was 31.8% However, as the relative tissue viability of this group was considerably higher than 50% this failure would not affect the overall classification of corrosivity. This acceptance criterion was therefore not fully satisfied and is reported as a deviation.

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

The test item was considered to be corrosive: UN GHS H314 Combination of sub-categories 1B and 1C. Category 1B has been indicated in interpretation of results and section 2.1 GHS classification in a precautionary approach.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is classified as skin corrosion, leading to classification as serious eye damage (Category 1)

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion/irritation:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viabilities for each treatment group were as follows:

Exposure period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	6.4	75.7
60 minute	100*	0.9	12.3

*The mean viability of the negative control tissues is set at 100%.

The quality criteria required for acceptance of results in the test were satisfied with the exceptionthat the Coefficient of Variation of the test item treated 3 minute exposure group exceeded the 30% limit. This is reported as a deviation.

The test item was considered to be corrosive: UN GHS H314 Combination of sub-categories 1B and 1C.

 

Eye irritation:

As the test substance is found to be corrosive to the skin, no eye irritation study is performed.

Justification for classification or non-classification

Based on the available data and the criteria of the CLP Regulation the substance is classified as corrosive to the skin, category 1B.

Therefore, the substance is also classified for serious eye damage 1, based on the criteria of the CLP Regulation.