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Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to the OECD guideline 431 and GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
N-(3-Aminopropyl)diethanolamine
IUPAC Name:
N-(3-Aminopropyl)diethanolamine
Constituent 2
Chemical structure
Reference substance name:
N-(3-aminopropyl)iminodiethanol
EC Number:
225-642-0
EC Name:
N-(3-aminopropyl)iminodiethanol
Cas Number:
4985-85-7
Molecular formula:
C7H18N2O2
IUPAC Name:
2-[(3-aminopropyl)(2-hydroxyethyl)amino]ethan-1-ol
Constituent 3
Reference substance name:
APDEA
IUPAC Name:
APDEA
Test material form:
other: pale green liquid
Details on test material:
- Name of test material (as cited in study report):
- Physical state: liquid
- Analytical purity: 86.4%
- Purity test date: 07 February 2011
- Lot/batch No.:A203E4010101
- Expiration date of the lot/batch: 07 February 2013
- Stability under test conditions: no data
- Storage condition of test material: room temperature in the dark

Test animals

Species:
other: Episkin: reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen
Details on test animals or test system and environmental conditions:
not applicable

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: 0.9% w/v sodium chloride solution was used as the negative control. Glacial acetic acid was used as the positive control.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µl
- Concentration (if solution): undiluted
Duration of treatment / exposure:
3, 60 and 240 minutes
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
EPISKINTM Model Kit 0.38 cm2
Supplier : SkinEthic Laboratories, Nice, France
Date received : 18 December 2012

Assessment of Direct Test Item Reduction of MTT
MTT Dye Metabolism, Cell Viability Assay
The MTT assay, a colourimetric 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.

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 2.2 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at room temperature for 3 hours. Untreated MTT solution was used as a control.

Pre-Incubation (Day 0: tissue arrival)
2.2 ml of maintenance medium, warmed to approximately 37ºC, was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12 well plate was used for each test item, control and time point. The tissues were incubated at 37ºC, 5% CO2 in air overnight. After 24 hours the medium underneath the tissues was refreshed and the tissues were returned to the incubator for a further 24 hours.
Since the test item was considered to have the ability to directly reduce MTT, water killed tissues were required. Water-killed tissues were prepared by placing untreated EPISKINTM tissues in a 12 well plate containing 2 ml of sterile distilled water in each well. The tissues were incubated at 37ºC, 5% CO2 in air for 48 hours ± 1 hour. At the end of the incubation period the water was discarded. Once killed, the tissues were stored in a freezer (-14 to -30ºC). Before use the tissues were thawed by placing each tissue in 2 ml of maintenance medium for 1 hour at room temperature.

Main Test
Application of Test Item and Rinsing (Day 2)
2.2 ml of assay medium, warmed to approximately 37ºC, was pipetted into 2 wells of the second and third columns of the 12 well plate. The tissues were transferred into the second column.
Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes.

At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.
2.0 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12 well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 hours ±5 minutes at 37ºC, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was taken using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 ml micro tubes containing 500 µl of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT loaded tissues.

Absorbance/Optical Density Measurements (Day 3)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 µl samples were transferred to the appropriate wells of a pre labelled 96 well plate. 200 µl of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vivo

Results
Irritation parameter:
other: relative mean viability of the test item treated tissues
Basis:
mean
Time point:
other: 3min
Score:
75.4
Remarks on result:
other: Not corrosive
Irritant / corrosive response data:
Test Item, Positive Control Item and Negative Control Item
Following quantitative correction of the results the relative mean viability of the test item treated tissues was:
240 minutes exposure : 5.0% (This result is considered inconclusive due to significant direct reduction of MTT)
60 minutes exposure : 32.4% (This result is considered inconclusive due to significant direct reduction of MTT)
3 minutes exposure : 75.4%

Quality Criteria
The relative mean tissue viability for the positive control treated tissues was 4.1% relative to the negative control treated tissues following the 240 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
The mean OD540 for the negative control treated tissues was 0.967. The negative control acceptance criterion was therefore satisfied.
Other effects:
The MTT solution containing the test item turned blue. This was taken to indicate the test item reduced MTT.
The direct reduction by the test item relative to the negative control value was:
3 minutes exposure : 16.6%
60 minutes exposure : 41.7%
240 minutes exposure : 53.7%
Direct reduction following the 3-Minute exposure was acceptable (<30% relative to the negative control). Direct reduction following the 60 minute and 240 minute exposures was greater than 30% relative to the negative control, 41.7% and 53.7% respectively. The results obtained from the 60 minute and 240 minute exposure period will therefore be considered inconclusive.

Applicant's summary and conclusion

Interpretation of results:
other: not a corrosive substance of category 1A
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
N-(3-Aminopropyl)diethanolamine was found to have caused significant direct reduction of MTT (>30% relative to the negative control) after 240 and 60 minutes exposure and therefore no conclusion can be drawn for these exposure times. After a 3 minutes exposure, the mean viability of 75.4% indicates that N-(3-Aminopropyl)diethanolamine is not a corrosive substance of category 1A.
Executive summary:

The purpose of this test is to evaluate the corrosivity potential of N-(3-Aminopropyl)diethanolamine using the EPISKINTM in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to be compatible with the OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004), the method B.40bis of Commission Regulation (EC) No 440/2008.

The EPISKINTMmodel is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) test items. 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. To identify this possible interference, N-(3-Aminopropyl)diethanolamine was checked for the ability to directly reduce MTT. The result indicated that N-(3-Aminopropyl)diethanolamine is able to directly reduced MTT and therefore the MTT viability assay was performed in parallel on viable and water-killed (non-viable) tissues in order to quantitatively correct the results of the viable tissues. Duplicate tissues were treated with N-(3-Aminopropyl)diethanolamine for exposure periods of 3, 60 and 240 minutes. Since N-(3-Aminopropyl)diethanolamine was considered to have the ability to directly reduce MTT, a water-killed (non-viable) tissue was also treated with the test item for each exposure period. 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 a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.  At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 540 nm (OD540). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). N-(3-Aminopropyl)diethanolamine was shown to directly reduce MTT and water-killed tissues were employed, the results of the MTT assay were therefore corrected. If direct reduction by the test item was greater than 30% of the negative control value, as shown by the water-killed (non-viable) tissues, additional steps must be taken into account or the test item may be considered incompatible with this test system. The direct reduction by N-(3-Aminopropyl)diethanolamine relative to the negative control value was:

240 minutes exposure

:

53.7%

60 minutes exposure

:

41.7%

3 minutes exposure

:

16.6%

Direct reduction following the 3-Minute exposure was acceptable (<30% relative to the negative control). Direct reduction following the 60 minute and 240 minute exposures was greater than 30% relative to the negative control, 41.7% and 53.7% respectively. The results obtained from the 60 minute and 240 minute exposure period will therefore be considered inconclusive.

  

Following quantitative correction of the results the relative mean viability of the test item treated tissues was:

240 minutes exposure

:

5.0% This result is considered inconclusive due to significant direct reduction of MTT

60 minutes exposure

:

32.4% This result is considered inconclusive due to significant direct reduction of MTT

3 minutes exposure

:

75.4%

The quality criteria required for acceptance of results in the test were satisfied.

N-(3-Aminopropyl)diethanolamine was found to have caused significant direct reduction of MTT (>30% relative to the negative control) after 240 & 60 minutes exposure and therefore no conclusion can be drawn for these exposure times. After a 3 minutes exposure, the mean viability of 75.4% indicates that N-(3-Aminopropyl)diethanolamine is not a corrosive substance of category 1A.

 

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