Potassium isobutyrate

Endpoint summary

Description of key information

Key studies are available for both skin and eye irritation. Studies are performed in accordance with an appropriate guideline and under the conditions of GLP.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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

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)

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

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

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

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 December 2018

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:

Adopted 09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 181101001
- Expiration date of the lot/batch: 31 October 2020
- Purity test date: not known


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (20 ± 5 °C); Keep away from humidity
- Stability under test conditions: H2O: 96h; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
- Solubility and stability of the test substance in the solvent/vehicle: H2O: > 1 g/L; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid: The test item is a neat surfactant (information was provided by the sponsor). It was tested at a concentration of 10% in Hank’s Balanced Salt Solution (HBSS).

FORM AS APPLIED IN THE TEST (if different from that of starting material) : liquid

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test.
- Number of animals: Not specified
- Characteristics of donor animals (e.g. age, sex, weight): The cattle were between 12 and 60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour and 10 minutes.
- Time interval prior to initiating testing: not specified
- indication of any existing defects or lesions in ocular tissue samples: none known
- Indication of any antibiotics used: no

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 10%

VEHICLE
HBSS: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10), batch no.: 20181220
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:

Exposure time of the controls and test item on the corneas was 10 minutes at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 hours at 32 ± 1 °C

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the initial incubation, the medium was completely changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used.

NUMBER OF REPLICATES
For each treatment group (negative control solution, test item and positive control), three replicates were used.

NEGATIVE CONTROL USED
HBSS: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10), batch no.: 20181220


POSITIVE CONTROL USED
Dimethylformamide, DMF, CAS-No. 68-12-2, undiluted, batch no.: 475235719

APPLICATION DOSE AND EXPOSURE TIME
After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative control solution, 750 µL test item and 750 µL positive control were applied to each replicate to the epithelial side of the cornea. Exposure time of the controls and test item on the corneas was 10 minutes at 32 ± 1 °C.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes. After thorough rinsing the anterior chamber with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red and the corneaholder were stored for additional 2 hours at 32 ± 1 °C (post-incubation).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
see above.

- POST-EXPOSURE INCUBATION:
2 hours.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean IVIS (3 values)

Value:

0.59

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The mean IVIS of the negative control has to show an IVIS ≤ 3.

- Acceptance criteria met for positive control:
According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean

- Range of historical values if different from the ones specified in the test guideline: Values for negative and positive controls were within the range of historical data of the test facility. Results were attached to the study report.

Opacity and permeability values:

The illuminance (unit: LUX) values which were measured before and after exposure are given in the following table:

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
(I) Measured values before exposure	1017	1016	1050	1058	1040	1032	1024	1030	1026
(I) Measured values after exposure	999	973	1024	1034	986	1002	291	311	315

Rep. = Replicate

The values in the following tables present the calculated opacity values, according to evaluation.

Opacity values negative control:

Parameter	Negative Control
	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	3.22	3.26	1.88
Opacity after exposure	3.98	5.14	2.92
Opacity Difference	0.77	1.89	1.05
Mean Opacity Difference	1.23

Opacity values test item and positive control:

Parameter	Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	1.56	2.27	2.60	2.92	2.68	2.84
Opacity after exposure	2.52	4.56	3.85	109.58	100.00	98.23
Opacity Difference	0.95	2.28	1.26	106.66	97.32	95.39
Opacity Difference corrected	-0.28	1.05	0.02	105.42	96.09	94.15
Mean Opacity Difference corrected	0.26			98.56

For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables:

Optical density at 492 nm of Blank

Parameter	cMEM without phenol red
1. Measurement	0.047
2. Measurement	0.049
3. Measurement	0.047
Mean	0.048

Optical density at 492 nm of Negative Control, Test Item and Positive Control

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
1. Measure-ment	0.055	0.051	0.051	0.055	0.052	0.114	0.680	2.040	2.001
2. Measure-ment	0.056	0.050	0.048	0.052	0.052	0.116	0.655	2.038	1.941
3. Measure-ment	0.054	0.050	0.050	0.053	0.052	0.116	0.655	2.001	1.922
1. Measure-ment – blank	0.0073	0.0033	0.0033	0.0073	0.0043	0.0663	0.6323	1.9923	1.9533
2. Measure-ment – blank	0.0083	0.0023	0.0003	0.0043	0.0043	0.0683	0.6073	1.9903	1.8933
3. Measure-ment – blank	0.0063	0.0023	0.0023	0.0053	0.0043	0.0683	0.6073	1.9533	1.8743
Mean of each replicate	0.0073	0.0027	0.0020	0.0057	0.0043	0.0677	0.6157	1.9787	1.9070
Mean of the 3 replicates	0.0040			--			--
Corrected	--	--	--	0.0017	0.0003	0.0637	3.0743*	1.9747	1.9030
Corrected mean of the 3 replicates	--			0.0219			2.3173

Rep. = Replicate

* Note: One value for the positive control was obtained by measurement of a fivefold diluted solution and multiplication of the absorbances with factor 5.

 

IVIS Values

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

      IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	0.88	1.29	42.98%
	1.93
	1.08
Test Item Potassium isobutyrate	- 0.26	0.59	124.53%
	1.05
	0.98
Positive Control DMF undiluted	151.54	133.32	11.89%
	125.71
	122.70

 

Note: the high relative standard deviation of the IVIS of test item is due to mathematical reasons, as the respective means are very small.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Potassium isobutyrate showed no effects on the cornea of the bovine eye. The calculated mean IVIS (In Vitro Irritancy Score) was 0.59 .According to OECD Guideline no. 437 (Oct. 2017) and in accordance with Regulation (EC) No.1272/2008 (EU CLP), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.
 

Executive summary:

Title of Study: Evaluation of Potassium isobutyrate in the Bovine Corneal Opacity and Permeability (BCOP) Test Method following OECD Guideline 437 and EU Method B.47

 

Findings and Results:

 

One valid experiment was performed.

Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old.

The test item Potassium isobutyrate was applied onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was tested at a concentration of 10% and it was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated mean IVIS (In VitroIrritancy Score) was 1.29.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated mean IVIS was 133.32.

 

Under the conditions of this study, the test item Potassium isobutyrate showed no effects on the cornea of the bovine eye. The calculated mean IVIS was 0.59.

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Eye irritation:

The test item Potassium isobutyrate showed no effects on the cornea of the bovine eye. The calculated mean IVIS (In Vitro Irritancy Score) was 0.59 .According to OECD Guideline no. 437 (Oct. 2017) and in accordance with Regulation (EC) No.1272/2008 (EU CLP), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.

Skin irritation:

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.