Potassium isobutyrate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Jan 2019 - 18 Jan 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

Version / remarks:

adopted 06. Jul. 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Potassium isobutyrate

EC Number:

243-077-8

EC Name:

Potassium isobutyrate

Cas Number:

19455-20-0

Molecular formula:

C4H8O2.K

IUPAC Name:

potassium 2-methylpropanoate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor / 181101001
- Expiration date of the lot/batch: 31. Oct. 2020
- Purity test date: 16 Jan 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 ± 5 °C); Keep away from humidity. The test item was stored in the test facility in a closed vessel at room temperature (20±5°C), protected from humidity.
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle:

Stability in solvents: H2O: 96h; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility: H2O: > 1 g/L; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

other: human

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM / EPI-200-SIT
- Tissue batch number(s): 28679
- Delivery date: 15. Jan. 2018
- Date of initiation of testing: 19 Jan 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 42 hours and 25 minutes
- Spectrophotometer: plate spectrophometer
- Wavelength: 570 nm


NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive or irritant to the skin if the tissue viability is less than or equal to 50% of the negative control.
- The test substance is considered to be non-irritant to skin if tissue viability is greater than 50% of the negative control tissue viability

Control samples:

yes, concurrent no treatment

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Tissue 1: 25.7 mg
Tissue 2: 26.4 mg
Tissue 3: 25.9 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS buffer

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.

Duration of treatment / exposure:

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.

Duration of post-treatment incubation (if applicable):

Tissues were set in the incubator for 23 hours and 30 minutes hours at 37 ± 1°C and 5.0 ± 0.5% CO2.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (3 tissues)

Value:

54.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The validity of the skin irritation study at LAUS GmbH was demonstrated in a proficiency study. For this purpose, 10 proficiency chemicals (indicated by the OECD 439 guideline) were tested.
All of the 10 proficiency chemicals were correctly categorized. Therefore, the proficiency of the skin irritation study was demonstrated.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Findings and Results

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 1. Absorbance values blank isopropanol (OD 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.034	0.034	0.037	0.037	0.035	0.04	0.035	0.035	0.035

 

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

Table 2. Absorbance Values negative control, test item and positive control (OD 570 nm)

Designation	Measurement	Negative Control	Potassium isobutyrate	Positive Control
Tissue 1	1	1.727	0.885	0.080
	2	1.696	0.874	0.080
Tissue 2	1	1.706	0.910	0.075
	2	1.698	0.899	0.075
Tissue 3	1	1.626	1.012	0.080
	2	1.617	1.002	0.079

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in table 9.1-a. The mean of the three tissues was also calculated.

Table 3. Mean Absorbance Values

Designation	Negative Control	Potassium isobutyrate	Positive Control
Mean – blank (tissue 1)	1.677	0.845	0.045
Mean – blank (tissue 2)	1.667	0.870	0.040
Mean – blank (tissue 3)	1.587	0.972	0.045
Mean of the three tissues	1.644	0.896	0.043

 

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 4. % Tissue Viability

Designation	Potassium isobutyrate	Positive Control
% Tissue viability (tissue 1)	51.4%	2.7%
% Tissue viability (tissue 2)	52.9%	2.4%
% Tissue viability (tissue 3)	59.1%	2.7%
% Tissue viability (mean)	54.5%	2.6%
± SD of mean tissue viability (%)	4.1%	0.2%

 

Assessment and Validity

Skin Irritation Potential of the Test Item

The mean value of relative tissue viability of the test item was reduced to 54.5 % after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as non-irritant to skin.

Validity and Acceptability

Validity criteria and results are stated in the following table:

Table 5. Validity

Criterion	Demanded	Found
OD of negative control	≥0.8 and≤ 2.8	1.6
% tissue viability of positive control SDS	£20% of negative control	2.6%
SD of mean viability of the tissue replicates (%)	≤18%	3.0% (negative control) 0.2% (positive control) 4.1% (test item)

 

All validity criteria were met.

Values for negative control and for positive control were within the range of historical data of the test facility.

 

Therefore, the experiment is considered valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Potassium isobutyrate is considered as non-irritant to skin.
After the treatment, the mean value of relative tissue viability was reduced to 54.5%. This value is above the threshold for skin irritation (50%). Therefore the substance is not considered to require classification for irritancy in accordance with Regulation (EC) No. 1272/2008.

Executive summary:

Findings and Results:

One valid experiment was performed.

Three tissues of the human skin model EpiDermTM were treated with the test item for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

DPBS-buffer was used as negative control and 5% SDS solution was used as positive con-trol.

After treatment with the negative control, the mean absorbance value was within the re-quired acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.6.

The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.6% (required: less than or equal to 20%).

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 54.5 %. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin.

Therefore, the test item Potassium isobutyrate is considered non- irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.