Potassium propionate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-01-07 to 2021-04-xx

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Potassium propionate

EC Number:

206-323-5

EC Name:

Potassium propionate

Cas Number:

327-62-8

Molecular formula:

C3H6O2.K

IUPAC Name:

potassium propionate

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Remarks:

moistened with 5 µL Milli_Q water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™ EPISKIN-SM™, 0.38 cm2
- Tissue batch number(s): Batch no.: 21EKIN002

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 15±0.5 min at room temperature
- Temperature of post-treatment incubation (if applicable): 42±1 h at 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed.
- Observable damage in the tissue due to washing: no


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3mg/mL
- Incubation time: 3h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: SDS concentration, MTT test: 1.5 mg/mL ≤ IC50 ≥ 3.0 mg/mL; result: 1.9 mg/mL
- Barrier function:
- Morphology:HES stained paraffin section: Multi-layered differentiated epidermis consisting of organized basal, spinous and granular layers, and a multilayered stratum corneum; result: Satisfactory;
Number of cell layers ≥ 4; result: 7.5 cell layers
- Contamination: Absence of HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B antigens HBs were verfied in the donors blood.


NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of -0.0023 and 0.0001, respectively. Therefore it was concluded that the test item did not induce color interference. In addition, because no color change was observed in the presence of MTT it was concluded that the test item did not interact with the MTT endpoint.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One experiment with 3 tissues per test item and controls were used.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 21.1 to 29.1 mg moistened with 5µL Milli-Q water


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL SDS
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 ± 0.5 min at room temperature

Duration of post-treatment incubation (if applicable):

42 ± 1 h at 37°C post incubation

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

#1

Value:

101

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No



ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, see any other information on results incl. tables
- Acceptance criteria met for positive control: Yes, see any other information on results incl. tables
- Acceptance criteria met for variability between replicate measurements: Yes, see any other information on results incl. tables
- Range of historical values if different from the ones specified in the test guideline:
see any other information on results incl. tables

Any other information on results incl. tables

Individual measurements of OD at 570 nm:

	A	B	C
	(OD570)	(OD570)	(OD570)
Negative control
OD570 measurement 1	1.3021	1.1217	1.1602
OD570 measurement 2	1.3011	1.1538	1.2347
Test item
OD570 measurement 1	1.2175	1.1848	1.2185
OD570 measurement 2	1.2863	1.2353	1.2070
Positive control
OD570 measurement 1	0.0729	0.1694	0.0892
OD570 measurement 2	0.0720	0.1732	0.0927

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Historical control data for In Vitro Skin Irritation Studies:

	Negative control (absorption; OD570)	Positive control (absorption; OD570)
Range	0.507 – 1.426	0.026 – 0.549
Mean	1.020	0.108
SD	0.155	0.087
n	147	147

SD = Standard deviation

n = Number of observations

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Potassium Propionate (100% a.i.) was not irritating in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted June, 26, 2020), Potassium propinate (100% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 15 minutes in triplicates. 5 μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 20 - 30 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

After 15 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (PBS) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment with Potassium Propionate compared to the negative control tissues was 101%. Since the mean relative tissue viability for the test substance was above 50%, Potassium Propionate is identified to be not irritating.