N-butyl 4-oxopentanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 19th to 23rd, 2018

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 28. July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Principles of method if other than guideline:

Additional information was taken from:
- 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 Function Test” (Altern Lab Anim. 2007 Dec; 35 (6): 559-601).
- Protocol for In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT), Rev. 07/11/2014, MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, Bratislava - Slovakia

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-butyl 4-oxopentanoate

EC Number:

218-143-4

EC Name:

N-butyl 4-oxopentanoate

Cas Number:

2052-15-5

Molecular formula:

C9H16O3

IUPAC Name:

butyl 4-oxopentanoate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM tissue kit
- Tissue batch number(s): 25887
- Delivery date: 20/03/2018
- The EpiDermTM tissue consists of 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 representing main lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.

SOLUTIONS AND MEDIA
- MTT solution: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (=MTT), which can be reduced to a blue formazan by the viable cells. A MTT stock solution of 5 mg/ml in DPBS buffer was prepared and stored in aliquots of 2 ml in the freezer (– 20 ± 5 °C). 2 ml of the stock solution were thawed and diluted with 8 ml of medium. This MTT-solution with the resulting concentration of 1 mg/ml was used in the test. For the pre-test (testing the ability of direct MTT reduction), the stock solution was thawed and diluted with serum-free MEM directly before use. For the main test, the stock solution was thawed and diluted with assay medium directly before use.
- MEM with Phenol Red for Pre-Test: Serum-free MEM (Minimum Essential Medium)
- Assay Medium: Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium)
- Isopropanol: used as extracting solvent for formazan
- DPBS-buffer: Solution for the rinsing of the tissues and solvent for MTT concentrate, also used as negative control. Composition of the subset used as negative control and for rinsing the test item from the tissues: KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 7H2O 2.16 g, H2O ad 1 litre. Composition of the subset used as solvent for MTT concentrate and for rinsing the outside of the inserts at the end of the incubation time with MTT: KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 2H2O 1.44 g, H2O ad 1 litre.

PRE-TESTS
- Nylon mesh compatibility: the test item was tested for possible reaction with the nylon mesh, which is used to ensure sufficient contact with the tissue surface. 30 µl of the test item were pipetted onto a nylon mesh on a microscope slide. No reaction with the nylon mesh was visible after an exposure time of 1 hour.
- Assessment of Coloured or Staining Test Items: it was tested whether the test item develops a colour without MTT addition. 30 µl test item were given in a test tube with 0.3 ml H2O demin. and incubated at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour. The resulting solution was colourless, therefore no binding capacity had to be tested.
- Assessment of Direct Reduction of MTT by the Test Item: the test item was tested for the ability of direct MTT reduction. To test for this ability, 30 µl test item were added to 1 ml of MTT solution and the mixture was incubated in the dark at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour. Untreated MTT medium was used as control. The MTT solution did not change its colour within 1 hour. Therefore, direct MTT reduction by the test item had not taken place and no data correction was necessary.

MAIN TEST
- Pre-incubation of tissues: all working steps were performed under sterile conditions. For each treatment group (negative control, test item and positive control) a 6-well-plate was prepared with 0.9 ml assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Then, the tissues were transferred into the wells, which contain medium by using sterile forceps and placed into the incubator at 37 ± 1°C and 5 ± 0.5% CO2 for 1 hour. After 1 hour pre-incubation, the other 3 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 ± 1 °C and 5.0 ± 0.5 % CO2 for 18 hours and 55 minutes for overnight pre-incubation.
- Treatement: one plate (3 tissues) was used as negative control; each tissue was treated with 30 µl DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tis-sue surface. One plate (3 tissues) was used as positive control; each tissue was treated with 30 µl 5 % SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface. One plate (3 tissues) 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 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1 °C and 5.0 ± 0.5 % CO2. 1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals. After thoroughly rinsing with DPBS, each tissue was blotted with sterile cellulose tissue, carefully dried with a sterile cotton tipped swab and then transferred into a new 6-well-plate with fresh assay medium (0.9 ml). Then, the tissues were set in the incubator for 23 hours and 25 minutes at 37 ± 1 °C and 5.0 ± 0.5 % CO2.
- Medium renewal: after post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 ml assay medium were filled in the lower row of the 6-well-plate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 hours and 15 minutes for post-incubation at 37 ± 1 °C and 5.0 ± 0.5 % CO2.
- MTT Assay: after a total incubation time of 42 hours and 40 minutes, a 24-well-plate was prepared with 300 µl freshly prepared MTT-solution (1 mg/ml) 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 at 37 ± 1°C and 5.0 ± 0.5% CO2. After this time, the MTT-solution 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 (120 rpm) for 2 hours at room temperature. After 2 hours, the inserts 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 spectrophotometer at 570 nm.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant/corrosive to skin if the viability after the exposure is less than or equal to 50 %
- The test substance is considered to be non-irritant to skin if the viability after the exposure is greater than 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 μl

NEGATIVE CONTROL
- Amount(s) applied: 30 μl

POSITIVE CONTROL
- Amount(s) applied: 30 μl

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 hrs and 40 min

Number of replicates:

three

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

30

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. OD of negative control: 1.3
- Acceptance criteria met for positive control: yes. % tissue viability of positive control SDS: 2.5 %
- Acceptance criteria met for variability between replicate measurements: 11.0 % (negative control), 0.0 % (positive control), 12.0 % (test item)

Any other information on results incl. tables

Measured Values

As blank, the optical density of isopropanol was measured in 8 wells of the 96-well-plate. The measured values and their mean are given in the following table:

Table: Absorbance values blank isopropanol (OD 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.039	0.038	0.040	0.040	0.039	0.038	0.040	0.039	0.039

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

Table: Absorbance Values negative control, test item and positive control (OD 570 nm)

Designation	Measurement	Negative Control	Test substance	Positive Control
Tissue 1	1	1.314	0.533	0.072
	2	1.484	0.556	0.072
Tissue 2	1	1.512	0.255	0.071
	2	1.477	0.255	0.073
Tissue 3	1	1.187	0.502	0.071
	2	1.230	0.525	0.071

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol. The mean of the three tissues was also calculated.

Table: Mean Absorbance Values

Designation	Negative Control	Positive Control	Test substance
Mean – blank (Tissue 1)	1.360	0.506	0.033
Mean – blank (Tissue 2)	1.456	0.216	0.033
Mean – blank (Tissue 3)	1.170	0.475	0.032
Mean of the three tissues	1.329	0.399	0.033

Comparison of Tissue Viability

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

Table: % Tissue Viability

Designation	Test substance	Positive Control
% Viability (Tissue 1)	38.1 %	2.5 %
% Viability (Tissue 2)	16.3 %	2.5 %
% Viability (Tissue 3)	35.7 %	2.4 %
% Viability Mean	30.0 %	0.0 %

Applicant's summary and conclusion

Interpretation of results:

other: The substance should be classified according to the CLP Regulation (EC) No.1272/2008 in Category 1 or Category 2

Conclusions:

The substance is a skin irritant (Cat.2) or a skin corrosive (Cat.1)

Executive summary:

The skin irritation potential of the substance was evaluated in the in-vitro test according to the OECD Guideline 439. Three tissues of the human skin model EpiDerm were treated with the substance for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size. DPBS-buffer was used as negative control and 5 % SDS solution was used as positive control. After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.3. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.5 %. The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18 %).

 

After the treatment with the test item, the mean value of relative tissue viability was reduced to 30.0 %. This value is below the threshold for skin irritation potential (50 %). The substance is considered at least irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.