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Toxicological information

Skin irritation / corrosion

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
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 material

Constituent 1
Chemical structure
Reference substance name:
2-[bis(2-ethylhexyl)amino]ethanol
EC Number:
202-912-6
EC Name:
2-[bis(2-ethylhexyl)amino]ethanol
Cas Number:
101-07-5
Molecular formula:
C18H39NO
IUPAC Name:
2-[bis(2-ethylhexyl)amino]ethan-1-ol
Details on test material:
Test Material Name: ETHOX 4256
Chemical Name: 2-(Bis(2-ethylhexyl)amino)ethanol
Lot #: 26D647

In vitro test system

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.

Results and discussion

In vitro

Resultsopen allclose all
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

Applicant's summary and conclusion

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).