Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-08-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Data obtained from a guideline study according to OECD Guideline 439 and therefore considered reliable without restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Acetamide
EC Number:
200-473-5
EC Name:
Acetamide
Cas Number:
60-35-5
Molecular formula:
C2H5NO
IUPAC Name:
acetamide
Test material form:
solid: crystalline
Details on test material:
Name Acetamid
Appearance colorless crystal substance
CAS No. 60-35-5
EINECS-No. 200-473-5
Molecular formula C2H5NO
Molecular weight 59.07 g/mol
Purity Pure substance > 99 %

Test animals

Species:
other: in vitro
Strain:
other: in vitro

Test system

Type of coverage:
open
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
Tissue 1: 26.5 mg
Tissue 2: 27.3 mg
Tissue 3: 26.1 mg
Duration of treatment / exposure:
60 minutes
Number of animals:
3 tissues
Details on study design:
Pre-Tests
First, it was tested whether the test item develops a colour without MTT addition. 24.7 mg 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 Acetamid was tested for the ability of direct formazan reduction. To test for this ability, 24.3 mg were added to 1 mL of MTT solution and the mixture was incubated 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, direct MTT reduction had not taken place, and no data correction was necessary.

Pre-Incubation 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 clean bench 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.

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. The tissues were wetted with 25 µL DPBS buffer before applying the test item and spreading it to match the tissue size.

Tissues were dosed in one minute intervals. After dosing the last tissue, all plates are transferred into the incubator for 35 min.. 60 min. 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: 11:23 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: 12:23 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 will be destroyed after finalisation of the final report.

MTT Assay
After a total incubation time of 42 ± 2 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 DPBS 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 pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for two hours at 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.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: % Formazan production
Value:
104.9
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60 min. Max. score: 50.0. Reversibility: other: not applicable. (migrated information)

Any other information on results incl. tables

From the measured absorptions, the mean of each tissue was calculated, subtracting the mean absorption of isopropanol. Mean and relative standard deviation (comparison of the three tissues) were also calculated.

Mean Absorption Values

Designation

Negative Control

Acetamid

Positive Control

Mean – blank (Tissue 1)

2.039

2.251

0.045

Mean – blank (Tissue 2)

2.167

2.095

0.047

Mean – blank (Tissue 3) 

1.882

2.038

0.043

Mean of the three Tissues

2.029

2.128

0.045

Relative Standard Deviation
of the three tissues

7.0%

5.2%

4.4%

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

% Formazan Production

Designation

Acetamid

Positive Control

% Formazan production (Tissue 1)

110.9%

2.2%

% Formazan production (Tissue 2)

103.3%

2.3%

% Formazan production (Tissue 3)

100.4%

2.1%

% Formazan production Mean

104.9%

2.2%

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The skin Irritation Potential of Acetamid was determined in the Human Skin Model Test following EU-Method B.46 resp. OECD 439.
Three tissues of the human skin model EpiDermTM were treated with Acetamid for 60 minutes.After the treatment with the test item, the relative absorbance values increased compared to the negative control to 104.9 %. This value is well above the threshold for irritation po-tential (50 %).

Therefore, Acetamid is considered as not skin irritant in the Human Skin Model Test.
Executive summary:

The skin Irritation Potential of Acetamid was determined in the Human Skin Model Test following EU-Method B.46 resp. OECD 439.

Three tissues of the human skin model EpiDermTMwere treated withAcetamidfor 60 minutes.

In average, 26.6 mg of the solid test item (wetted with 25 µL DPBS-buffer) 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 0.8 ≤ mean OD ≤ 2.8. The positive control showed clear irritating effects. Variation within tissues was acceptable (< 18%).

After the treatment with the test item, the relative absorbance values increased compared to the negative control to 104.9 %. This value is well above the threshold for irritation potential (50 %).

 

Therefore, Acetamid is considered as not skin irritant in the Human Skin Model Test.