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Diss Factsheets

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
yes
Remarks:
Coefficient of Variation between tissue replicates treated with the test item for 3-min and 1-h exceeded the allowed limit of 30%. Evaluation: Since all individual viabilities were in the same sub-category, the test outcome was considered valid.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(bis(2-(1,3-dimethylbutylideneamino)ethyl)amino)-3-phenoxypropan-2-ol
EC Number:
271-340-7
EC Name:
1-(bis(2-(1,3-dimethylbutylideneamino)ethyl)amino)-3-phenoxypropan-2-ol
Cas Number:
68541-07-1
Molecular formula:
C25H43N3O2
IUPAC Name:
1-(bis(2-(1,3-dimethylbutylideneamino)ethyl)amino)-3-phenoxypropan-2-ol
Test material form:
liquid
Specific details on test material used for the study:
Identification: 1-(bis(2-(1,3-dimethylbutylideneamino)ethyl)amino)-3-phenoxypropan-2-ol
Appearance: Light yellow liquid
Batch: UL18401660
Purity/Composition: ca. 91.48%
Test item storage: At room temperature
Stable under storage conditions until: 15 April 2020 (expiry date)

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
not specified
Details on animal used as source of test system:
The model consists of normal, human-derived epidermal keratinocytes which have been
cultured to form a multilayered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers, and a multi-layered stratum
corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those
found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate
membranes of 10 mm cell culture inserts.

Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
Test system
EpiDerm Skin Model (EPI-200, Lot no.: 28864 Kit H, Kit I and Kit J).
The model consists of normal, human-derived epidermal keratinocytes which have been
cultured to form a multilayered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers, and a multi-layered stratum
corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those
found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate
membranes of 10 mm cell culture inserts.

Experimental Design
Test for the Interference of the Test Item with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly
reduce MTT. The cell viability measurement is affected only if the test item is present on the
tissues when the MTT viability test is performed.

Test for Color Interference by the Test Item
The test item was checked for possible color interference before the study was started. Some
non-colored test items may change into colored items in aqueous conditions and thus stain the
skin tissues during the 1-hour exposure. To assess the color interference, 50 µL of the test
item or 50 µL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The
mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the
exposure time the mixture was shaken and it was checked if a blue / purple color change was
observed.

Test for Reduction of MTT by the Test Item
The test item was checked for possible direct MTT reduction before the study was started. To
assess the ability of the test item to reduce MTT, 50 µL of the test item or 50 µL Milli-Q
water as a negative control were added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands)
solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for
approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a
blue / purple color change or a blue / purple precipitate was observed.

Test System Set Up
Tissues
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the
next day, at least one hour before starting the assay the tissues were transferred to 6-well
plates with 0.9 mL DMEM.

Freeze-killed tissues (EPI-200, Lot no.: 28855 Kit M, Appendix 4)
Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to
(≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues
were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 mL
DMEM. Further use of killed tissues was similar to living tissues.

DMEM (Dulbecco’s Modified Eagle’s Medium)
Supplemented DMEM, serum-free supplied by MatTek Corporation.

MTT medium
MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both
supplied by MatTek Corporation.

Environmental conditions
All incubations, with the exception of the test item incubation of 3 minutes at room
temperature, were carried out in a controlled environment, in which optimal conditions were a
humid atmosphere of 80 - 100% (actual range 67 - 83%), containing 5.0 ± 0.5% CO2
in air in
the dark at 37.0 ± 1.0°C (actual range 36.2 - 37.3°C). Temperature and humidity were
continuously monitored throughout the experiment. The CO2
percentage was monitored once
on each working day. Temporary deviations from the temperature, humidity and CO2
percentage may occur due to opening and closing of the incubator door. Based on laboratory
historical data these deviations are considered not to affect the study integrity.

Test Item Preparation
No correction was made for the purity/composition of the test item.
The liquid test item was applied undiluted (50 µL) directly on top of the tissue.

Application/Treatment of the Test Item
The skin tissues were kept in the refrigerator the day they were received. The next day, at
least 1 hour before the assay was started the tissues were transferred to 6-well plates
containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue (see
figure 1). The plates were incubated for approximately 1 hour at 37.0 ± 1.0ºC. The medium
was replaced with fresh DMEM just before the test item was applied. The test was performed
on a total of 4 tissues per test item together with a negative control and positive control. Two
tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.
Fifty µL of the undiluted test item was added into the 6-well plates on top of the skin tissues.
In addition, since the test item reacted with the MTT medium, two freeze-killed tissues were
treated with test item and two freeze-killed non treated tissues were used per exposure time
for the cytotoxicity evaluation with MTT.
For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water
(negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both
the 3-minute and 1-hour time point.
After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen
Corporation, Breda, The Netherlands) to remove residual test item. The skin inserts were
carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues
(= one application time) were dosed and rinsed.
The first test was rejected since the %NSMTT was too high (not reported). To this end the
test was repeated.

Cell Viability Measurement
The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at
37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and
formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room
temperature. The amount of extracted formazan was determined spectrophotometrically at
570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50µl per tissue
Duration of treatment / exposure:
3 minutes and 1 hour
Duration of post-treatment incubation (if applicable):
not applicable
Number of replicates:
2 per time point

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes
Value:
ca. 36
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour
Value:
ca. 24
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was
within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance
limit <=2.8) and the laboratory historical control data range (See Appendix 3). The mean
relative tissue viability following the 1-hour exposure to the positive control was 8.3%.
In the range of 20 - 100% viability the Coefficient of Variation between replicates of the
negative control tissues was <= 15%, indicating that the test system functioned properly.
The Coefficient of Variation between replicates of the test item tissues was >30% (47% and
50% for the 3-minute and 1-hour procedure, respectively, which is above the 30% acceptability criteria.
Evaluation: Since all individual viabilities were in the same sub-category, the test outcome was considered valid.

Any other information on results incl. tables

The test item was checked for color interference in aqueous conditions and possible direct

MTT reduction by adding the test item to MTT medium.  Because a color change was

observed by adding MTT-medium it was concluded that the test item did interact with the

MTT endpoint.  The non-specific reduction of MTT by the test item was -1.77% and 6.56%

of the negative control tissues for the 3-minute and 1-hour treatment, respectively.  The net

OD of the treated killed tissues was subtracted from the ODs of the test item treated viable

tissues.

The mean absorption at 570 nm measured after treatment with the test item and controls are

presented in Table 1 below.

Table 2 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with

the test item compared to the negative control tissues.  Skin corrosion is expressed as the

remaining cell viability after exposure to the test item.  The relative mean tissue viability

obtained after the 3-minute and 1-hour treatments with the test item compared to the negative

control tissues was 36% and 24% respectively.  Because the mean relative tissue viability for

the test item was below 50% after 3 minute treatment it is considered to be corrosive.  

Table 1

Mean Absorption in the in vitro Skin Corrosion Test with the test item

 

 3 minute application

 1-hour application

A (OD570)

B (OD570)

Mean (OD570)

SD

A (OD570)

B (OD570)

Mean (OD570)

SD

Negative control

1.456

1.704

1.580

0.175

1.728

1.921

1.824

0.137

Test Item

0.755

0.399

0.577

0.252

0.298

0.590

0.444

0.207

Positive control

0.137

0.128

0.132

0.006

0.113

0.191

0.152

0.055

SD = Standard deviation

Duplicate exposures are indicated by A and B.

(1)The test item values are corrected for the non-specific MTT reaction (6.56% at the 1 hour treatment).

In this table the values are corrected for background absorption (0.0429).  Isopropanol was

used to measure the background absorption.

Table 2

Mean Tissue Viability in the in vitro Skin Corrosion Test with the test item

 

 3-minute application

viability (percentage of control)

1-hour application

viability (percentage of control)

 

 Negative control

 100

 100

 the test item

 36

 24

 Positive control  8.4  8.3

Table 3  

Coefficient of Variation between Tissue Replicates

   3 minute  1 hour
 Negative control  15  10
 Test Item  36  24
 Negative control  6.7  41

Applicant's summary and conclusion

Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
In conclusion, the test item is corrosive in the in vitro skin corrosion test under the
experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate 1-(bis(2-(1,3-dimethylbutylideneamino)

ethyl)amino)-3-phenoxypropan-2-ol for its ability to induce skin corrosion on a human three

dimensional epidermal model (EpiDerm (EPI-200)).  The possible corrosive potential of the

test item was tested through topical application for 3 minutes and 1 hour.

Batch UL18401660 of the test item was a light yellow liquid.  The test item was applied

undiluted (50 µL) was applied directly on top of the skin tissue.  

The positive control had a mean relative tissue viability of 8.3% after the 1-hour exposure.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was

within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance

limit <=2.8) and the laboratory historical control data range.  In the range of 20 - 100%

viability the Coefficient of Variation between replicates of the negative control tissues was  

<= 15%, indicating that the test system functioned properly.  The Coefficient of Variation

between replicates of the test item tissues was >30% (47% and 50% for the 3-minute and

1-hour procedure, respectively) (see Study plan deviation).  

Because a color change was observed by adding MTT-medium it was concluded that the test

item did interact with the MTT endpoint.  The non-specific reduction of MTT by the test item

was -1.77% and 6.56% of the negative control tissues for the 3-minute and 1-hour treatment,

respectively.  The net OD of the treated killed tissues was subtracted from the ODs of the test

item treated viable tissues.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item.  The

relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item

compared to the negative control tissues was 36% and 24%, respectively.  Because the mean

relative tissue viability for the test item was below 50% after the 3-minute treatment it is

considered to be corrosive.  

In conclusion, the test item is corrosive in the in vitro skin corrosion test under the

experimental conditions described in this report.