Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27.03.2013-22.04.2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully documented study run under GLP and according to international guidelines

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Qualifier:
according to guideline
Guideline:
other: • ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Func-tion Test (Altern Lab Anim. 2007 Dec; 35 (6): 559-601).
Qualifier:
according to guideline
Guideline:
other: • Protocol for IN VITRO EpiDermTM SKIN IRRITATION TEST (EPI-200-SIT), Rev. 3/23/2009, MatTek Corporation, Ashland, MA 01721, USA
Principles of method if other than guideline:
This in-vitro study was performed in order to evaluate the potential of Ethylamylketon (5-methylheptan-3-one, CASNr: 541-85-5) to evoke skin irritation in a human-skin-model.
The test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT (3-[4,5-dimethyl thiazole 2-yl] 2,5-diphenyl-tetrazoliumbromide), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
5-methylheptan-3-one
EC Number:
208-793-7
EC Name:
5-methylheptan-3-one
Cas Number:
541-85-5
Molecular formula:
C8H16O
IUPAC Name:
5-methylheptan-3-one
Details on test material:
-Name of the test material: Ethylamylketon (5-methylheptan-3-one, CASNr: 541-85-5)
-Batch no./Ref. No.: 12023305
-Expiration date: 31. May 2013
-Storage: store in a tightly closed container, cool and dry, in a well ventilated place

Test animals

Species:
other: human-derived epidermal keratinocytes (EPI-200 tissues)
Strain:
other: Commercially available Epi-200-SIT-Kit
Details on test animals or test system and environmental conditions:
The EpiDermTM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, 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 EpiDermTM tissues are cultured on specially prepared cell culture inserts.
Epi-200 tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava (Batch: 18311).
Preincubation of tissues:
Eight 6-well-plates were prepared with 0.9 mL assay medium in three of the six wells (up-per row). The tissues were inspected for viability. Viable tissues were transferred (three per plate) in the wells with the medium using sterile forceps under the laminar air flow bank and placed into the incubator at 37 °C and 5 % CO2 for one hour.
After the pre-incubation (one hour), the other three wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 °C and 5 % CO2 for 18 hours.

Test system

Type of coverage:
other: nylon mesh
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
Amount / concentration applied:
Applied volume: 30 uL of test item
Duration of treatment / exposure:
60 minutes
Details on study design:
Pre-Tests:
First, it was tested whether the test item develops a colour without MTT addition. 30 µL were given in a test tube with 0.3 mL H2O demin. and incubated at 37 °C and 5 % CO2 for 60 minutes. The resulting solution was colourless, therefore no binding capacity had to be tested.
Then, the test item Ethylamylketon was tested for the ability of direct formazan reduction. To test for this ability, 30 µL were added to 1 mL of MTT solution and the mixture was in-cubated in the dark at 37 °C and 5 % CO2 for 60 minutes. Untreated MTT solution was used as control. The MTT solution didn’t change its colour within one hour, therefore, di-rect MTT reduction had not taken place, and no data correction was necessary.
Finally, Ethylamylketon was tested for possible reaction with the nylon mesh which is used to ensure sufficient contact with the tissue surface. 30 µL Ethylamylketon were brought onto a nylon mesh on a microscope slide. No reaction with the mesh was visible after 1 h incubation at 37 °C.

Treatment
The pre-incubated tissues were placed into fresh 6-well-plates containing 0.9 mL assay medium per well, using the upper row only.
One plate (three tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer. One plate was used as positive control; each tissue was treated with 30 µL SDS-solution. One plate was used for treatment with the test item. 30 µL test item were applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.

Tissues were dosed in one minute intervals. After dosing the last tissue, all plates are transferred into the incubator for 35 minutes. 60 minutes after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in one-minute-intervals.
Dosing of first tissue: 11:00 h
Dosing of last tissue: 11:15 h
Start of incubation at 37 °C: 11:25 h
End of incubation at 37 °C: 12:00 h
Rinsing of first tissue: 12:00 h
Rinsing of last tissue: 12:15 h
After rinsing, each tissue was dried with a sterile cotton tip and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). The tissue surfaces were evaluated visually under the stereo microscope, excess test item was removed, where necessary.
Then the tissues were set in the incubator for 24 hours.

Medium Renewal
For three incubated tissues, a new 6-well-plate with 0.9 mL assay medium in the upper row was prepared. The tissues were removed from the incubator and shaken for ten min (500 rpm). Then the inserts were transferred into the new 6-well-plate and set into the in-cubator for 18 ± 2 hours for post-incubation.
The medium from the “old” 6-well-plates was collected in the labelled 24-well-plate. It can be stored for 12 months at – 20 °C for possible interleukin analysis. As the result of the test was unambiguous, the samples were destroyed after finalisation of the final report.

MTT Assay
After a total incubation time of 42 hours, a 24-well-plate was prepared with 300 µL freshly prepared MTT-reagent in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours ± 5 min.
After this time, the MTT reagent was aspirated and replaced by PBS buffer. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipet-ted, taking care to reach the upper rim of the insert. The plate was then shaked for two hours room temperature.
After two hours, the inserts in which formazan had been produced were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in a plate spectral photometer at 570 nm.

Calculations
The photometric absorption of the negative controls is considered as 100 %. For each replicate of test item and positive control, formazan production is calculated as % photometric absorption compared with the mean of the negative controls:
%Formazan production=(OD1replicate test item resp.positve control/ ODmean of negative controls)*100 
1OD= Optical Density

Validation criteria:
Skin irritation potential of the test item is assessed as given in the following table:
Table8.2-a      Assessment of Irritation Potential
% Formazan production
Assessment
=50 % of negative control
Irritant
> 50 % of negative control
Non-irritant
 

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: % Formazan production
Value:
6
Remarks on result:
other:
Remarks:
Basis: mean. Max. score: 100.0. Reversibility: not reversible. (migrated information)

Any other information on results incl. tables

Measured Values

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:

Table1.1-a      Absorption values blank isopropanol (OD at 570 nm)

Replicate

1

2

3

4

5

6

7

8

Mean

Absorption

0.040

0.056

0.043

0.037

0.039

0.042

0.038

0.034

0.041

 

The absorption values of negative control, test item and positive control are given in the following table:

Table1.1-b      Absorption values negative control, test item and positive control (OD at 570 nm)

Designation

Measurement

Negative Control

Ethylamylketon

Positive Control

Tissue 1 

1

2.292

0.178

0.100

2

2.245

0.182

0.102

Tissue 2 

1

2.179

0.180

0.120

2

2.219

0.179

0.119

Tissue 3 

1

2.055

0.151

0.116

2

2.081

0.148

0.115

 

From the measured absorptions, the mean of each tissue was calculated, subtracting the mean absorption of isopropanol as given in table 1.1-a. Mean and relative standard deviation (comparison of the three tissues) were also calculated.

Table1.1-c      Mean Absorption Values

Designation

Negative Control

Ethylamylketon

Positive Control

Mean – blank (Tissue 1)

2.228

0.139

0.060

Mean – blank (Tissue 2)

2.158

0.139

0.079

Mean – blank (Tissue 3) 

2.027

0.109

0.075

Mean of the three Tissues

2.138

0.129

0.071

Relative Standard Deviation
of the three tissues

4.8 %

13.4 %

14.1 %

 


Comparison of Formazan Production

For the test item and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

Table1.2-a      % Formazan Production

Designation

Ethylamylketon

Positive Control

% Formazan production (Tissue 1)

6.5 %

2.8 %

% Formazan production (Tissue 2)

6.5 %

3.7 %

% Formazan production (Tissue 3)

5.1 %

3.5 %

% Formazan production Mean

6.0 %

3.3 %

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item Ethylamylketon (5-methylheptan-3-one, CASNr: 541-85-5) is considered irritant.
Executive summary:

Three tissues of the human skin model EpiDermTMwere treated with Ethylamylketon (5-methylheptan-3-one, CASNr: 541-85-5) for 60 minutes.

30 µL of the liquid test item (using a nylon mesh) were applied to each tissue and spread to match the tissue size (0.63 cm2; as indicated by supplier). DPBS-buffer was used as negative control, 5 % SDS-solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control showed clear irritating effects. Variation within tissues was acceptable (< 18 %). After the treatment with the test item, the relative absorbance values were reduced to 6.0 %. This value is well below the threshold for irritation potential (50 %).

Therefore, Ethylamylketon (5-methylheptan-3-one, CASNr: 541-85-5) is considered as irritant in the Human Skin Model Test.