2-[bis(2-ethylhexyl)amino]ethanol

Endpoint summary

Description of key information

In vitro Skin corrosion and Skin irritation study

In vitro Eye irritation study

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:

2017

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)

Deviations:

no

GLP compliance:

no

Remarks:

This is a non-GLP study, based on OECD test guideline 431 (In vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method).

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Cultured human derived epidermal keratinocytes, MatTek Corporation

Vehicle:

unchanged (no vehicle)

Details on test system:

Upon receipt, the EpiDerm tissue transwell discs were stored at 2-8 ºC and used within 48 hours of receipt from the supplier. On the day prior to testing, each EpiDerm tissue transwell disc was inspected for air bubbles between the agarose gel and Millicell® insert prior to opening the sealed package. Tissue discs with air bubbles greater than 50% of the Millicell® area were not used for testing. An appropriate number of EpiDerm tissues were aseptically removed from the 24-well shipping plate and transferred to a 6-well plate containing pre-warmed assay medium. The EpiDerm tissues were then incubated at approximately 37ºC in a humidified atmosphere of approximately 5% CO2 for 18 ± 3 hours to acclimate the tissue prior to treatment.

On the day of treatment, the EpiDerm tissues were transferred to new 6-well plates containing 0.9 mL/well of fresh pre-warmed assay medium and exposed to the test material (50 µL), in conjunction with negative (water) and positive controls (8N Potassium hydroxide solution (KOH)) for two exposure periods: 3 and 60 minutes. The 3 minute exposure groups were held at room temperature during the treatment incubation, while the 60 minute exposure groups were placed in the incubator at standard culture conditions (approximately 37ºC with 5% CO2/95% air) during treatment. Following the exposure period, the tissues were rinsed with sterile DPBS (approximately 20 times) to remove test substance (extra rinses were implemented as necessary). Following rinsing, tissue inserts were evaluated for cell viability using MTT assay. Each tissue insert was transferred to a well containing 300 µL MTT (1mg/mL) solution in a 24-well plate and tissues were incubated for 3 ± 0.1 hr at standard cell culture conditions. After incubation the tissues were washed with DPBS and formazan was extracted in 2 mL extractant solution (isopropanol) overnight at room temperature. The extract solution was mixed and 2 x 200 µL aliquots were transferred to the appropriate wells of a 96-well plate. The amount of extracted formazan was measured spectrophotometrically at 570 nm (OD570) with a Microplate Reader.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates (3 minute)

Value:

107.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates (60 minutes)

Value:

119.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

In the corrosion assay, the positive control (KOH) reduced the relative mean tissue viability to 19.8% and 11.2%, following 3 and 60 minute exposures, respectively. Similarly, in the irritation assay, the positive control (Triton X-100) reduced relative mean tissue viability to 6.1% following 60 minute exposure. The positive control results demonstrated appropriate study conduct and tissue responsiveness.
The mean tissue viabilities of the test material-dosed tissues following the 3 minute exposure period was 107.3% and following the one hour exposure period was 119.9%. Since the mean tissue viability was > 50% at the 3 minute exposure and >15% at the 60 minute exposure, the test material was interpreted as negative for corrosion potential in the EpiDerm corrosion assay (UN GHS Non-Corrosive).

Executive summary:

The test substance was evaluated for skin corrosion and irritation potential inin vitroEpiDerm skin corrosion and irritation assays (MatTek Corporation; Ashland, MA). The EpiDerm tissue model consists of normal, human-derived epidermal keratinocytes that are cultured to form a stratified, squamous epithelium similar to that found in human epidermis tissue.

To assess corrosion potential, the test material was topically applied to the EpiDerm tissue for 3 and 60 minutes. In this study, sterile water and 8N potassium hydroxide (KOH) served as the negative and positive controls, respectively. At the end of exposure, cell viability in treated and control tissues were measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The test substance is considered corrosive (UN GHS Cat. 1) if the relative mean viability is ≤ 50% at three minutes or ≥ 50% at three minutes but less than 15% at one hour.

In the corrosion assay, the mean tissue viability of the positive control (KOH) following the 3 minute and 60 minute exposures were 19.8% and 11.2%, respectively, thereby demonstrating appropriate assay responsiveness. The mean tissue viabilities of the test material-dosed tissues following the 3 minute and 60 minute exposures were 107.3% and 119.9%, respectively. Therefore, under these conditions, the test material was interpreted as negative for corrosion potential in the EpiDerm corrosion assay (UN GHS Non-Corrosive).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Remarks:

This is a non-GLP study, based on OECD 439 test guideline (In Vitro Skin Irritation: Reconstructed Human Epidermis (RHE) Test Method).

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Cultured human derived epidermal keratinocytes, MatTek Corporation

Vehicle:

unchanged (no vehicle)

Details on test system:

Upon receipt, the EpiDerm tissue transwell discs were stored at 2-8 ºC and used within 48 hours of receipt from the supplier. On the day prior to testing, each EpiDerm tissue transwell disc was inspected for air bubbles between the agarose gel and Millicell® insert prior to opening the sealed package. Tissue discs with air bubbles greater than 50% of the Millicell® area were not used for testing. An appropriate number of EpiDerm tissues were aseptically removed from the 24-well shipping plate and transferred to a 6-well plate containing pre-warmed assay medium. The EpiDerm tissues were then incubated at approximately 37ºC in a humidified atmosphere of approximately 5% CO2 for 18 ± 3 hours to acclimate the tissue prior to treatment.

On the day of treatment, all EpiDerm tissues were transferred to new 6-well plates containing 0.9 mL/well of fresh pre-warmed assay medium and 50 µL liquids of test material in conjunction with negative (DPBS) and positive controls (1% TRITON™ X-100) was dispensed atop the tissue. After dosing, the tissue plates were incubated for 35 ± 1 minutes at standard culture conditions. Following incubation, the plates were transferred to the laminar flow hood and incubated at room temperature for 25 minutes. Following the total of 60 ± 1 minutes treatment period, the tissues were rinsed with sterile DPBS to remove test substance (extra rinses were implemented as necessary). Following rinsing, the tissue inserts were transferred to new 6-well plates containing 0.9 mL/well of fresh pre-warmed assay medium and incubated at standard culture conditions for an initial post-treatment incubation of 24±2 hours. After the initial post-treatment incubation, the inserts were transferred into clean wells of 6 well plates pre-filled with 0.9 mL fresh pre-warmed assay medium and further incubated for additional 18±2 hours (total post-treatment incubation period (recovery): 42 ± 2 hours).
Following post-treatment incubation, tissue inserts were evaluated for cell viability using MTT assay. Each tissue insert was transferred to a well containing 300 µL MTT solution in a 24-well plate and tissues were incubated for 3 ± 0.1 hr at standard cell culture conditions. After incubation the tissues were washed with DPBS and formazan was extracted in 2 mL extractant solution overnight at room temperature. The extract solution was mixed and 2 x 200 µL aliquots were transferred to the appropriate wells of a 96-well plate. The amount of extracted formazan was measured spectrophotometrically at 570 nm (OD570) with a Microplate Reader.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

51.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

55.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

55.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

54.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

The mean relative tissue viability for EpiDerm tissues treated with the test material and positive control (1% TRITON™ X-100) were 54.4% and 6.1%, respectively.
According to the EpiDerm skin irritancy prediction model, a test substance is considered an irritant to skin if the mean tissue viability is ≤ 50%. Therefore, based on the present EpiDerm study results, the test material was interpreted to be a non-irritant (UN GHS Cat NC) to skin.

Executive summary:

The test substance was evaluated for skin corrosion and irritation potential inin vitroEpiDerm skin corrosion and irritation assays (MatTek Corporation; Ashland, MA). The EpiDerm tissue model consists of normal, human-derived epidermal keratinocytes that are cultured to form a stratified, squamous epithelium similar to that found in human epidermis tissue.

To assess irritation potential, the test substance was topically applied to the EpiDerm tissue for 60 minutes, followed by a 42-hour post-exposure recovery. In this irritation study, Dulbecco’s Phosphate Buffered Saline (DPBS) and 1% TRITON™ X-100served as the negative and positive controls, respectively. Following recovery, the cell viability was measured in treated and control tissues using the MTT assay and the data reported as a percentage of the mean of negative control.

A test chemical was considered to possess skin irritation potential (UN GHS Cat 2) if the relative cell viability was less than or equal to 50% compared to negative control. 

In the irritation assay, the mean relative cell viability of the positive control 1% Triton X-100) was 6.1%, thereby demonstrating appropriate assay responsiveness. The mean tissue viability of the test substance treated tissues were 54.4% (i.e.≤50%). Therefore, under these conditions ETHOX 4256was interpreted as negative for dermal irritation potential(UN GHS No Category)in theEpiDerm irritation assay.

Therefore, under the conditions of the EpiDerm corrosion and irritation studies, the test substabce predicted to be negative for dermal irritation potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This is a non-GLP study and is based on OECD TG 492

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test Material Name: ETHOX 4256
Chemical Name: 2-(Bis(2-ethylhexyl)amino)ethanol
Lot/Reference/Batch Number: 26D647
Purity/Characterization (Method of Analysis and Reference): The non-GLP certificate of analysis lists the purity as 99.80% POE (1) DI 2 EH AMINE by gas chromatography (Tucker, 2016).
Stability: The test material ETHOX 4256, Lot 26D647, was not tested for neat test material stability.

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcular three-dimensional model has been extensively characterized and currently has an OECD test guideline (OECD TG 492) for identifying chemicals not requiring classification and labeling for eye irritation or serious eye damage.
The EpiOcular model estimates the potential ocular irritation of a test substance by measuring cytotoxicity following topical exposure (Freeman et al., 2010) (MatTek Corporation, Ashland, MA). This assay assumes that in vitro cytotoxicity is directly proportional to in vivo damage that a test substance would inflict upon exposure to the eye (cornea) (Jackson et al., 2006). This assumption is based in part on the Maurer (2002) proposed hypothesis, which suggests that the level of ocular irritation is related to the extent of initial injury, regardless of the processes leading to tissue damage.

Principle of the Test System:
The EpiOcular model (model number OCL-200) uses Normal Human Epidermal Keratinocytes (NHEK) from a single donor as the cell source. The cells are cultured on polycarbonate membranes of cell culture inserts (MILLICELLs, 10 mm diameter,
0.6 cm² surface), in serum-free medium to form a multilayered (5-8 cell layers), highly differentiated stratified, squamous epithelia that closely mimics human eye (corneal) epithelium at biochemical and physiological levels. The EpiOcular tissue is mitotically and metabolically active and releases many of the pro-inflammatory agents (cytokines) that are important in ocular irritation and inflammation (Klausner et al., 2000).

Supplier and Location:
MatTek Corporation; Ashland, Massachusetts

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Preparation of the Test Material:
Test material was tested at a concentration of 100% (neat/as provided), following supplier’s protocol (MatTek Corporation).

Route of Administration:
The test material was administered by topical application to the ocular tissue.

Experiment Procedure:
Upon receipt, the EpiOcular tissue transwell discs were stored at 2-8ºC and used within 48 hours of receipt from the supplier. On the day of testing, an aliquot of 0.9 mL of EpiOcular assay medium (MatTek Corporation) was dispensed into the wells of 6-well plates. Each EpiOcular tissue disc was inspected for air bubbles between the agarose gel and Millicell insert prior to opening the sealed package. Cultures with air bubbles greater than 50% of the Millicell (transwell disc) area were not used for testing.
The EpiOcular tissues were incubated at approximately 37ºC in a humidified atmosphere of approximately 5% CO2 for 60 ± 5 min. At the end of the first pre-incubation period, the inserts were transferred into wells containing fresh, warm assay medium. The testing included treating the inserts with 50 μL (liquids) or 50 mg (solids), DPBS (negative control; 30±2 minutes exposure time), 0.3% TRITON™ X-100 (positive control; 30±2 minutes exposure time), or test material(s) (three exposure times; 2, 15, or 30 min).
Following the exposure periods, the EpiOcular tissues were carefully washed with DPBS (at least 5 times) to remove residual test substance. Following washes, the Millicell inserts were submerged in fresh assay media and incubated at 37ºC and 5% CO2 for approximately 12±2 minutes to remove any test chemical absorbed into the tissue. Subsequently, the Millicell inserts were further incubated in fresh medium for 120±15 minutes at standard culture conditions (post-exposure incubation). After incubation, the tissue inserts were transferred to a well containing 300 μL MTT solution in a 24-well plate and tissues were incubated for 3 ± 0.1 hr at standard cell culture conditions. Following incubation, the tissues were washed with DPBS and the MTT dye (formazan crystals) was solubilized and extracted from the inserts by incubating each insert in 2 mL of extract reagent (MatTek Corporation) overnight at room temperature. The extract solution was mixed and 2 x 200 μL aliquots of the extract solution was transferred to a 96-well plate and the optical density of the extracted formazan was quantified at 570 nm (OD570) using a Microplate Reader.

Duration of treatment / exposure:

The testing included treating the inserts with 50 μL (liquids) or 50 mg (solids), DPBS (negative control; 30±2 minutes exposure time), 0.3% TRITON™ X-100 (positive control; 30±2 minutes exposure time), or test material(s) (three exposure times; 2, 15, or 30 min).

Duration of post- treatment incubation (in vitro):

Following washes, the Millicell inserts were submerged in fresh assay media and incubated at 37ºC and 5% CO2 for approximately 12±2 minutes to remove any test chemical absorbed into the tissue. Subsequently, the Millicell inserts were further incubated in fresh medium for 120±15 minutes at standard culture conditions (post-exposure incubation).

Number of animals or in vitro replicates:

3 replicates per time-point and for positve and negative control.

Irritation parameter:

other: Mean Viability %

Run / experiment:

2 minute

Value:

109.9

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Mean Viability %

Run / experiment:

15 minute

Value:

103.6

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Mean Viability %

Run / experiment:

30 minute

Value:

90.5

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Assessment of Direct Test Chemical Reduction of MTT:
One limitation of this assay method is a possible interference of the test material with the MTT assay. A colored test substance or one that directly reduces MTT (and thereby mimics dehydrogenase activity of the cellular mitochondria) may interfere with the MTT end point. To assess potential direct test material reduction, 30 μL of ETHOX 4256 was incubated with 1 mL of the MTT reagent at standard cell culture conditions for 60 min. Untreated (no test material) MTT medium was used as the negative control. Results from this experiment suggested no direct reduction of MTT dye by ETHOX 4256, as the test material did not turn the MTT solution to a blue/purple color.

Criteria for Determination of a Valid Test:
The results for negative and positive controls met the following assay acceptance criteria, suggesting appropriate conduct of the study:
1) The corrected mean OD570 value of the negative control tissues (exposed for 60 minutes) was 2.009 (i.e., ≥ 1.00 criteria set by the tissue manufacturer).
2) The relative mean viability of the positive control (0.3% TRITON™ X-100) was 13.7% (i.e. < 50% compared to the negative control).

Text Table 1. Culture Viability of ETHOX 4256 in the EpiOcular Eye Irritation Model

Chemical Name	Treatment plus 120±15 Min Recovery			Mean Viability (%)
	Replicate 1	Replicate 2	Replicate 3
ETHOX 4256 2 minute	114.8	114.3	100.6	109.9
ETHOX 4256 15 minute	100.2	109.5	101.1	103.6
ETHOX 4256 30 minute	89.1	89.9	92.4	90.5
Negative Control*	99.3	102.6	98.1	100.0
Positive Control*	15.9	11.2	14.0	13.7

*Negative Control: DPBS; Positive Control: 0.3% TRITON™ X-100

Text Table 2. ET-40 of ETHOX 4256 in EpiOcular Eye Irritation Model

Chemical Name	ET-40 (min)	EpiOcular Classification (Neat)
ETHOX 4256	> 30.0	UN GHS Cat NC
Positive Control*	< 30.0	UN GHS Cat 1 or 2

*Positive Control: 0.3% TRITON™ X-100

Interpretation of results:

GHS criteria not met

Conclusions:

The mean relative cell viability of the positive control-treated tissue was 13.7% (i.e. ≤ 40%) and the ET-40 value of ETHOX 4256 was > 30 minutes (Text Tables 1 and 2). Therefore, under these conditions, ETHOX 4256 was interpreted as a potential ocular non-irritant (UN GHS Cat NC) in the EpiOcular assay.

Executive summary:

ETHOX 4256 was evaluated for eye irritation potential in an in vitro EpiOcular eye irritation assay (MatTek Corporation; Ashland, MA). The EpiOcular tissue model consists of normal, human-derived epidermal keratinocytes that are cultured to form a stratified, squamous epithelium similar to that found in the cornea. In this assay, ETHOX 4256 was topically applied to the EpiOcular tissue for 2, 15, or 30 minutes followed by a 120±15 minute postexposure recovery. Following recovery, the cell viability was measured in treated and control tissues using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and the data reported as a percentage of the mean of negative control. An ET-40 value was calculated for ETHOX 4256, which is the time of exposure that resulted in reduction in cell viability to 40% of negative control level. The test substance was considered a severe irritant (UN GHS Cat 1) or an irritant (UN GHS Cat 2) in the EpiOcular assay if the ET-40 was less than 3 or 30 minutes, respectively. The test substance was considered a non-irritant (UN GHS Cat NC) if the ET-40 was > 30 minutes. In this study, Dulbecco’s Phosphate Buffered Saline (DPBS) and 0.3% TRITON™ X-100 served as the negative and positive controls, respectively.

The mean relative cell viability of the positive control-treated tissue was 13.7% (i.e. ≤ 40%) and the ET-40 value of ETHOX 4256 was > 30 minutes. Therefore, under these conditions, ETHOX 4256 was interpreted as a potential ocular non-irritant (UN GHS Cat NC) in the EpiOcular assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An in vitro skin irritation and corrosion assay was performed on the test material. Both assays were negative leading to the conclusion that this substance is not irritating or corrosive to the skin

the In vitro eye irritation study was negative indicating this substance is not irritating to the eyes.

Justification for classification or non-classification

GHS criteria for classification as a skin irritant are not met.