Potassium propionate

Endpoint summary

Description of key information

study conducted according to OECD test guideline 431 (adopted 18 June 2019); an EpiSkin™Model was exposed to 25 mg Potassium propionate for 3 minutes and 1h, in both cases the viability of the used disks was > 50% and > 15%, respectively, criteria according to guideline were met, not classified

study conducted according to OECD guideline 439 (adopted 26 June 2020);; a Skin Ethic™ Model was exposed to 20-30 mg of Potassium Propionate for 15 min. The viability after a post-incubtaion period of 42h was > 50 % (101%), criteria according to guideline were met, not classified

 study conducted according to OECD TG 437 (26 June 2020) treatment of bovine corneas with a 20% (w/v) solution in physiological saline of Potassium Propionate for 4h; IVIS of 6, inconclusive.

Read-across approach using the OECD QSAR Toolbox, based on seven structurally similar substances protassium propionate is not irritaing to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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

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 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

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

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

 

 

 

 

 

 

 

 

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.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-21 to 2020-12-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Justification for test system used:

The corrosivity potential of a chemical may be predicted by measurement of its cytotoxic effect, as reflected in the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] assay, on the EpiDermTM Model (EPI-200-SCT) reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD No. 404) and is specifically approved as a replacement for the in vivo skin corrosivity test within OECD No. 431.

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:EpiDerm™ Model
- Tissue batch number(s): Batch No.: 30893
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 2020-09-24

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes at room temperature (23.4-23.6°C) and 1 hours at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: insert was filled and emptied 20 times in a constant soft stream of PBS in a glass beaker filled with at least 100 mL PBS solution
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg MTT / mL medium
- Incubation time: 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 3 hours
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT QC assay, 4 hours, n=3; Acceptance criteria: OD (540 - 570 nm) [1.0 - 3.0]; Result: 1.748 ± 0.215
- Barrier function: ET-50 assay, 100 µL 1% Triton X-100, 4 time-points, n=3, MTT assay; Acceptance criteria: ET-50 [4.77 - 8.72 hrs]; Results: 4.79 hrs
- Contamination:
HIV-1 virus - Oligonucleotide-directed amplification: Not detected
Hepatitis-B virus - Oligonucleotide-directed amplification: Not detected
Hepatitis-C virus - Oligonucleotide-directed amplification: Not detected
Bacteria, yeast, and other fungi - long term anitbiotic, antimycotic free culture: Not detected

NUMBER OF REPLICATE TISSUES: In this assay, two replicates for the test item were used. Two negative controls and two positive controls were also run. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific colour optical density (OD) evaluation.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not necessary

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg


NEGATIVE CONTROL
- Concentration (if solution): 50 µL

POSITIVE CONTROL
- Concentration (if solution): 50 µL (8M KOH)

Duration of treatment / exposure:

3 minutes at room temperature (23.4-23.6°C) and 1 hours at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere.

Number of replicates:

duplicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of two used disks

Value:

2.123

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: No


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the two negative control tissues should be 2 0.8 and 5 2.8 and negative control OD values should not be below historically established boundaries. Cirteria met, see below.
- Acceptance criteria met for positive control: The positive control treated tissues showed 27.6% (3 minutes exposure period) and 7.0% (1 hour exposure period) cell viability demonstrating the proper performance ofthe assay. Criteria met, see below.
- Acceptance criteria met for variability between replicate measurements: In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤30%. The Coefficient ofVariation ofviability between the two test item-treated tissue samples in the
MTT assay was 3.6% (3 minutes exposure period) and 4.9% (1 hour exposure period). The Coefficient of Variation of viability between the two negative control treated tissue samples in the MTT assay was 3.1% (3 minutes exposure period) and 0.7% (1 hour exposure period).
The Coefficient of Variation of viability between the two positive control treated tissue samples in the MTT assay was 11.2% (3 minutes exposure period) and 17.6% (1 hour exposure period). Criteria met.
- Range of historical values if different from the ones specified in the test guideline:
OD data of two replicate tissues at the negative control in this study at 3 minutes exposure period were higher (2.047 and 2.139) than the maximum OD of the historical control range (OD: 1.959).
OD data of one replicate tissue at the positive control in this study at 3 minutes exposure period was higher (0.624) than the maximum OD of the historical control range (OD: 0.542).
OD data of two replicate tissues at the negative control in this study at 1 hour exposure period were higher (2.197 and 2.174) than the maximum OD of the historical control range (OD: 1.984).
OD data of one replicate tissue at the positive control in this study at 1 hour exposure period was higher (0. 172) than the maximum OD of the historical control range (OD: 0.161).
The study data was considered to be close enough to the historical range, and within the range expected by the kit supplier, hence the Study Director considered that these control results have no impact on the results or integrity of the study since the positive control material showed clear positive result and the acceptable mean percentage viability ofthe tissue replicates exposed for 1-hour with the positive control is ≤ 15%.according to the OECD No. 431.

VALIDITY OF THE TEST
The mean OD value of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 and negative control OD values should not be below historically established boundaries. Mean viability of the tissue replicates exposed for 1-hour with the positive control is ≤ 15%.
In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
The mean OD value of the blank samples should be <0.1.

INTERPRETATION OF TEST RESULTS
A chemical is classified "corrosive" in any case:
if the relative tissue viability after 3-minute treatment with a test item is decreased below 50%.
and if the relative tissue viability after 1-hour treatment with a test item is decreased below 15% and after 3 minutes ≥ 50%.
A chemical is classified "non-corrosive" in any case:

if the relative tissue viability after 3-minute treatment with a test item is above 50% and 1 hour ≥ 15%

Historical data: 3 MINUTES EXPOSURE PERIOD

	Mean value (OD)	Standard deviation (SD)	Minimum value (OD)	Maximum value (OD)	Number of cases
Negative control
Year 2020	1.757	0.102	1.570	1.959	16
Positive control
Year 2020	0.358	0.140	0.066	0.542	16
Year 2020 viability %	20.3	8.0	3.9	33.2

Negative control: Distilled Water

Positive control: 8M Potassium hydroxide solution (8M KOH)

SD: Standard deviation

OD: Optical density (absorbance)

Note: All OD values (measured at 570) are background corrected values.

Historical data: 1 HOUR EXPOSURE PERIOD

	Mean value (OD)	Standard deviation (SD)	Minimum value (OD)	Maximum value (OD)	Number of cases
Negative control
Year 2020	1.702	0.151	1.374	1.984	16
Positive control
Year 2020	0.122	0.020	0.080	0.161	16
Year 2020 viability %	7.2	7.2	4.5	9.1

Negative control: Distilled Water

Positive control: 8M Potassium hydroxide solution (8M KOH)

SD: Standard deviation

OD: Optical density (absorbance)

Note: All OD values (measured at 570) are background corrected values.

 

Optical Density (OD) and the Calculated Relative Viability % of the Samples

3 minutes exposure period

Substance	Optical density (OD)			Viability (%RV)	CV (%)
		Measured	Blank corrected
Negative control: Distilled water	1 2	2.092 2.183	2.047 2.139	97.8 102.2	- -
	mean	-	2.093	100.0	3.1
Positive control: 8 M KOH	1 2	0.577 0.668	0.532 0.624	25.4 29.8	- -
	mean	-	0.578	27.6	11.2
Test Item	1 2	2.222 2.113	2.178 2.068	104.0 98.8	- -
	mean		2.123	101.4	3.6

Notes:

1. Mean blank value was 0.044.


2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).


3. CV = Coefficient of Variation (CV % = Standard Deviation / mean viability * 100)

 

Optical Density (OD) and the Calculated Relative Viability % of the Samples

1 hour exposure period

Substance	Optical density (OD)			Viability (%RV)	CV (%)
		Measured	Blank corrected
Negative control: Distilled water	1 2	2.241 2.218	2.197 2.174	100.5 99.7	- -
	mean	-	2.185	100.0	0.7
Positive control: 8 M KOH	1 2	0.179 0.217	0.134 0.172	6.1 7.9	- -
	mean	-	0.153	7.0	17.6
Test Item	1 2	1.929 2.063	1.884 2.018	86.2 92.4	- -
	mean	-	.951	89.3	4.9

Notes:

1. Mean blank value was 0.044.


2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).


3. CV = Coefficient of Variation (CV % = Standard Deviation / mean viability * 100)

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

In the present test conducted according to OECD test guideline 431 (adopted 18 June 2019) an EpiDerm™ Model (EPI-200-SCT) was exposed to Potassium propionate (25 mg) for 3 minutes and 1 h.
The mean cell viability after 3 minutes of treatment was 101.4%, the mean cell viability after 1 hour treatment was 89.3% compared to the negative control. This is above the threshold of 15 % after 1 hour and 50% after 3 minute therefore the test item was considered to be non- corrosive to skin under the conditions of this assay. The experiment met the validity criteria, and therefore the study was considered to be valid.
In conclusion, under the conditions of this in vitro EpiDerm™SCT Model corrosivity assay, the results indicate that Potassium propionate is non-corrosive.

Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 431 (In Vitro Skin Irritation) (adopted June 19, 2019), Potassium Propionate was applied to the EpiDerm human epidermis model tissue for an exposure period of 3 to 60 minutes in duplicates. 25μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 25 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

 

After 3 minutes exposure at room temperature and 1 hours at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 3h at 37°C with MTT solution. MTT solution (300 µL/well) was added to each well below the skin units (except the colour control units which were incubated in Assay Medium). 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 (8M Potassium hydroxide solution) and negative (deionised water) 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 3 minutes treatment with Potassium Propionate compared to the negative control tissues was 101.4%, after the 1 hour treatment period the treatment versus control value was 89.3%. Since the mean relative tissue viability for the test substance was above 50%, Potassium Propionate is identified to be not irritating.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation, other

Remarks:

read-across aproach as predicted with the OECD QSAR Toolbox

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Justification for type of information:

1. SOFTWARE
OECD QSAR Toolbox v4.4.1
2. MODEL (incl. version number)
OECD QSAR Toolbox v4.4.1
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS: 327-62-8
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: eye irritation
- Unambiguous algorithm: Read-across approach
- Defined domain of applicability: set boundaries, logKow, bioavailability, structural features
- Appropriate measures of goodness-of-fit and robustness and predictivity: please refer to 'Attached justification'
- Mechanistic interpretation: please refer to 'Attached justification'

5. APPLICABILITY DOMAIN
- Descriptor domain: logKow
- Structural domain: aliphatic acids, salt, contains Carbon and Oxygen, Monoconsitutent
- Mechanistic domain: biovailability, strucutral features causing an eye irritation alert
- Similarity with analogues in the training set: please refer to 'Attached justification'

6. ADEQUACY OF THE RESULT
The substance lies in the applicability domain as defined by catgorization, thus, the prediction is considered reliable.

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Software tool(s) used including version: OECD QSAR Toolbox v4.4.1
- Model(s) used: OECD QSAR Toolbox v4.4.1
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

no

Irritation parameter:

other: not applicable for in silico study

Run / experiment:

not applicable for in silico study

Vehicle controls validity:

other:

Remarks:

not applicable for in silico study

Negative controls validity:

other:

Remarks:

not applicable for in silico study

Positive controls validity:

other:

Remarks:

not applicable for in silico study

Remarks on result:

other: in silico prediction

Remarks:

Based on ReA Approach calculated with the OECD QSAR Toolbox v4.4.1, the substance is predicted not eye irritating.

Interpretation of results:

other: not irritating according to estimation from QSAR Toolbox

Conclusions:

Based on the performed Read-across to similar substances using the OECD QSAR Toolbox, potassium propionate is considered not irritating to the eye.

Executive summary:

A read-across approach was performed using the OECD QSAR Toolbox. For categorization of structurally similar substances first the OECD HPV Chemical Categories profiler was used focussing only aliphatic acids. The subcategorization was conducted with the Lipinski Rule Oasis, the Aquatic toxicity classification by ECOSAR, Substance type, and the Chemical elements profiler. These steps of subcategorization resulted in 7 structurally similar subbstances defining the applicability domain of this approach. Potassium propionate lies in all applicability domains of the used approach. From the seven similar substances three were not irritating to the eye and one was classified as GHS not met. Thus, the prediction of potassium propionate was not irritaing to the eye.

 

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-01-05 to 2021-04-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750µL
- Concentration (if solution): 20% (w/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:

240 ± 10 minutes for incubation with the test item and 90 ± 5 minutes for the treatment with Na-fluorescein

Number of animals or in vitro replicates:

three corneas per treatment

Details on study design:

NUMBER OF REPLICATES triplicate

NEGATIVE CONTROL USED yes physiological saline

POSITIVE CONTROL USED yes, 20% Imidazole solution

APPLICATION DOSE AND EXPOSURE TIME 750 µL of a 20% solution (either test item or Imidazole) were applied on the corneas for 240 ± 10 minutes

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least three washing steps

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: decision criteria as indicated in the TG were used.

Irritation parameter:

in vitro irritation score

Run / experiment:

treatment

Value:

6

Vehicle controls validity:

valid

Remarks:

IVIS: 1

Negative controls validity:

not valid

Positive controls validity:

valid

Remarks:

IVIS: 121

Interpretation of results:

study cannot be used for classification

Conclusions:

The present study conducted according to OECD TG 437 (26 June 2020) revealed that treatment of bovine corneas with a 20% (w/v) solution in physiological saline of Potassium Propionate for 4h resulted in an IVIS of 6. According to OECD TG 437 the IVIS of the substance was > 3 and < 55, thus, no prediction can be made.

Executive summary:

This in vitro study was performed to assess them corneal irritation and damage potential of Potassium Propionate (20% in physiological saline) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 26 June 2020.

The corneae were incubated with the test substance and controls for 240± 1 min. After rinsing with saline, the corneae were incubated for another 90 ± 5 min for treatment with Na-fluorescein. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

A 20% dilution of the test substance in physiological saline caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 6.

The positive control (20% (w/v) Imidazole) increased the opacity and permeability of the corneae in both experiments (mean in vitro irritation score 121).

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments (mean in vitro irritation score 1.0).

Since the mean in vitro irritancy score of the test substance was <55.1, a 20% dilution Potassium Propionate in physiological saline is considered to not be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

In a dermal irritation study performed in accordance with OECD Guideline 431 (In Vitro Skin Irritation) (adopted June 19, 2019), Potassium Propionate was applied to the EpiDerm human epidermis model tissue for an exposure period of 3 to 60 minutes in duplicates. 25μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 25 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

After 3 minutes exposure at room temperature and 1 hours at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 3h at 37°C with MTT solution. MTT solution (300 µL/well) was added to each well below the skin units (except the colour control units which were incubated in Assay Medium). 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 (8M Potassium hydroxide solution) and negative (deionised water) 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 3 minutes treatment with Potassium Propionate compared to the negative control tissues was 101.4%, after the 1 hour treatment period the treatment versus control value was 89.3%. Since the mean relative tissue viability for the test substance was above 50%, Potassium Propionate is identified to be not irritating.

 

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.

 

Eye irritation:

This in vitro study was performed to assess them corneal irritation and damage potential of Potassium Propionate (20% in physiological saline) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 26 June 2020.

The corneae were incubated with the test substance and controls for 240± 1 min. After rinsing with saline, the corneae were incubated for another 90 ± 5 min for treatment with Na-fluorescein. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

A 20% dilution of the test substance in physiological saline caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 6.

The positive control (20% (w/v) Imidazole) increased the opacity and permeability of the corneae in both experiments (mean in vitro irritation score 121).

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments (mean in vitro irritation score 1.0).

Since the mean in vitro irritancy score of the test substance was <55.1, a 20% dilution Potassium Propionate in physiological saline is considered to not be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

A read-across approach was performed using the OECD QSAR Toolbox. For categorization of structurally similar substances first the OECD HPV Chemical Categories profiler was used focussing only aliphatic acids. The subcategorization was conducted with the Lipinski Rule Oasis, the Aquatic toxicity classification by ECOSAR, Substance type, and the Chemical elements profiler. These steps of subcategorization resulted in 7 structurally similar subbstances defining the applicability domain of this approach. Potassium propionate lies in all applicability domains of the used approach. From the seven similar substances three were not irritating to the eye and one was classified as GHS not met. Thus, the prediction of potassium propionate was not irritaing to the eye.

 

 

Justification for classification or non-classification

Following exposure of the EpiDerm™Model (EPI-200-SCT) to the test item, Potassium Propionate, the mean cell viability after 3 minutes of treatment was 101.4%, the mean cell viability after 1 hour treatment was 89.3% compared to the negative control. This is above the threshold of 15% after 1 hour and 50% after 3 minute therefore the test item was considered to be non-corrosive to skin under the conditions of this assay. The experiment met the validity criteria, and therefore the study was considered to be valid.
In conclusion, under the conditions of this in vitro EpiDerm™SCT Model corrosivity assay, the results indicate that Potassium Propionate does not need to be classified according to Regulation (EC) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) with respect to skin corrosion.

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. 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.

 

Based on the presented data, Potassium Propionate does not need to be classified according to Regulation (EU) No. 1272 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) with respect to skin corrosion/irritation.

 

In an in vitro study to assess them corneal irritation and damage potential of Potassium Propionate (20% in physiological saline) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 26 June 2020, three corneae were incubated with the test substance and controls for 240± 1 min. After rinsing with saline, the corneae were incubated for another 90 ± 5 min for treatment with Na-fluorescein. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant. A 20% dilution of the test substance in physiological saline caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 6.

The positive control (20% (w/v) Imidazole) increased the opacity and permeability of the corneae in both experiments (mean in vitro irritation score 121).

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments (mean in vitro irritation score 1.0).

Since the mean in vitro irritancy score of the test substance was <55.1, a 20% dilution Potassium Propionate in physiological saline is considered to not be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

In order to evaluate whether potassium propionate causes irritation to the eye currently a second test for eye irritation is running. Due to the absence of any draft results and in order to provide a proper classification of the substance a read-across approach was performed with the OECD QSAR Toolbox. Based on structurally similarities and similar physicochemical properties seven substances for comparison were identified. Of these seven substances three were not irritant to the eye and one was classified as GHS criteria not met. Hence, and because potassium propionate lies in the applicability domain of the category boundaries, the result is considered reliable. Potassium propionate was predicted as not irritating to the eye.

Thus, the substance is not classified as Irritating to the eye according to Regulation (EU) No. 1272/2008 (CLP).