Potassium neodecanoate

Endpoint summary

Description of key information

Skin corrosion: not corrosive (OECD 435; GLP)

Skin irritation: irritating (OECD 439, GLP)

Eye irritation: corrosive/severe irritant to the eyes (OECD 437, GLP compliant)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-07 to 2018-03-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Version / remarks:

2015-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICCVAM: Corrositex: An in vitro test method for assessing dermal corrosivity potential of chemicals.

Version / remarks:

1996-06

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: < 30 °C, dry

Test system:

artificial membrane barrier model

Source species:

other: not specified

Cell type:

other: synthetic macromolecular bio-barrier

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

not applicable

Justification for test system used:

Corrositex TM is a validated and accepted in vitro method to assess if a test item can produce skin corrosion and to distinguish between GHS corrosivity categories 1A, 1B, and 1C.

Vehicle:

unchanged (no vehicle)

Details on test system:

SOURCE AND COMPOSITION OF MEMBRANE BARRIER USED
- Was the Corrositex® test kit used: yes (lot no. CT120516, supplier: Invitro International; Irvine)
- Components: the test kit contained BIOBARRIER matrix and diluent, vials filled with CDS, confirm reagent, qualify test tubes, one tray of membrane discs
- Apparatus and preparation procedures: the preparation was completed at least 2 hours prior to running tests. The entire content of the BIOBARRIER diluent was added to the vial of BIOBARRIER matrix powder. The vial was heated to 68°C (± 1°C) in a water bath under smooth agitation. After complete dissolution (approximately 20 min.) the solution was allowed to sit for 5 min. to allow any air bubbles to rise to the surface. 200 μL of the BIOBARRIER were pipetted into each membrane disc. The BIOBARRIERS were set on the tray and kept in the cold (2 - 8°C) for at least two hours.

WAS THE COMPATIBILITY TEST PERFORMED: yes
This step ensures that the sample is compatible with the CORROSITEX™ system. 100 mg of the test substance are added to the Qualify test tube. Solids were shaken to dissolve solids, if necessary. If the colour or consistency of the CDS changes at the sample/testing fluid interface, the test material is qualified for the assay. If no reaction is observed within five minutes, the sample is not qualified for the CORROSITEX™ Assay.

WAS THE TIMESCALE CATEGORY TEST PERFORMED: yes
This step established the category of cut-off times for the sample. 100 mg of the test substance were added to the tubes labeled Tube A and Tube B. After shaking a colour change was observed in either of the tubes and colour was matched to the corresponding colour charts on the CORROSITEX™ Testing Protocol Poster. Test materials having high acid/alkaline reserves are defined as Category 1 materials, while those with low acid/alkaline reserves are defined as Category 2 materials. If no colour change had been observed in either tube, CONFIRM reagent was added to Tube B. After shaking, the resulting colour was matched to the colour chart on the CORROSITEX™ Testing Protocol Poster. If the test item has a strong inherent colour or shows other characteristics impairing a clear categorization according to the colour chart, the pH value can be measured in tubes A and B and is used to confirm/determine the category of the test item, according to the Corrositex® Reference Manual.

TEMPERATURE USED DURING TREATMENT: room temperature

METHOD OF DETECTION
- Chemical or electrochemical detection system: chemical detection system

METHOD OF APPLICATION (CLASSIFICATION TEST):
The CDS vials were warmed to room temperature (17 - 25˚C) before using. Vials 1 - 4 were utilized for sample replicate testing (test item: 4 vials; negative control: 1 vial; positive control: 1 vial; colour reference for CDS: 1 vial). One BIOBARRIER disc was added on top of the first vial (discs were not longer in the vial than two minutes before adding the test samples). 500 mg of the test item were applied evenly on the top of the BIOBARRIER disc and starting time was recorded. This step was repeated for the remaining vials, staggering each start time by e.g. 10 seconds (but not longer than 2 minutes). The start time difference for each vial was subtracted from the final time to determine the net response time. As soon as a reaction had been observed, the time was recorded.

DATA ANALYSIS:
The test item was categorised according to the criteria in table 1 as presented in the field "Any other information on materials and methods incl. tables" below. For Category 1 substances, test chemicals will be categorized as non-corrosive in case no colour change occurs after 240 minutes. For Category 2 substances, test chemicals will be categorized as non-corrosive in case no colour change occurs after 60 minutes. The mean time of the four sample replicates to activate the CDS was calculated.

TEST ACCEPTANCE CRITERIA:
The test meets acceptance criteria if:
- Test item qualifies in qualification test
- Positive control activates CDS > 3 - 60 min.
- Negative control activates CDS not before 60 min.

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

other: colour reference for the chemical detection system

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 500 mg of the test item
- an application aid (nylon mesh) was used to achieve direct contact with the synthetic membrane. Compatibility of test material and nylon mesh was confirmed microscopically.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 500 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 500 µL

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

Test item: quadruplicates
Negative control: single measurement
Positive control: single measurement

Irritation / corrosion parameter:

penetration time (in minutes)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test substance showed no corrosive effects

Other effects / acceptance of results:

QUALIFICATION TEST
The test substance was compatible with the CORROSITEX™ Assay, as assessed in the qualification step. The categorization step and the classification step could be performed.

CATEGORISATION TEST
A direct colour change was not observed. CONFIRM reagent was added to tube B and the category was read from the CORROSITEX™ colour chart. The chemical has been categorized to category 2.

ACCEPTANCE OF RESULTS:
The test substance proved its ability to activate the CDS.

- Acceptance criteria met for negative control: the negative control did not activate the CDS before 60 min. (> 60 min.)

- Acceptance criteria met for positive control: the positive control activated the CDS between 3 - 60 min. (19.90 min.)

CLASSIFICATION TEST:

The mean time, required to activate the CDS was > 60min.

	CORROSITEX™ Time [min]	Colour Change	Consistency Change
Replicate 1	> 60	No	No
Replicate 2	> 60	No	No
Replicate 3	> 60	No	No
Replicate 4	> 60	No	No
Mean ± SD	> 60
Positive control	26.02	Yes	No
Negative control	> 60	No	No

Interpretation of results:

GHS criteria not met

Conclusions:

Potassium neodecanoate is not corrosive to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-15 to 2018-04-13

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:

2015-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012-07-06

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

2014-11-07

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry and cool

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human-derived epidermal keratinocytes

Cell source:

other: humans

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human-derived epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

other: Dulbecco's phosphate buffered saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT; MatTek)
- Tissue lot numbers: 25883 (one tissue was used in order to evaluate the applicability of testing), 25893 (used for the main experiment)

A pre-test was conducted on one single tissue in order to evaluate the applicability of testing:
Since the physical form of the test material was identified as being sticky, a pre-test on one single skin tissue was conducted in order to evaluate the feasibility of testing. The test material passed the pre-test as the test material could be applied evenly on the skin tissue and it was possible to wash off the complete test material of the skin tissue surface. Thus, the main experiment was performed.


TEST FOR DIRECT MTT REDUCTION
- to check the non-specific MTT-reducing capability of the test item 25 mg of the test item was mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator (5 % CO2, 95 % RH).
- untreated MTT medium was used as control.
- if the mixture turns blue/purple, the test item is presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using freeze-killed tissues.

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 25 mg of the test item was mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min (5 % CO2, 95 % RH).
- if the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential using additional living tissues treated with the test item.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 minutes followed by incubation at room temperature until the 60 ± 1 minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment period the tissues were washed 15 times with DPBS.
- subsequently, the inserts were submerged three times in DPBS and shaken to remove rests of the test item.
- then inserts were rinsed once from the inside and the outside with sterile DPBS.
- inserts were placed in prepared 6-well plates containing pre-warmed fresh assay medium per well.
- plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing fresh assay medium and incubated for additional 18 ± 2 h.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes
- Extraction of formazan: after the MTT incubation period, the tissues were rinsed three times with DPBS and allowed to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature for 2 hours with shaking on a plate shaker.
Before using the extracts, the plate was shaken for at least 15 minutes on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. Optical density (OD) was measured with a filter band without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [mean ODtest item / positive control / ODmean of negative control] * 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues were calculated and used for classification according to the following prediction model:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues. The test item is considered to be Corrosive or irritant to skin in accordance with regulation EC 1272/2008 (UN GHS "Category 1" or “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin in accordance with regulation EC 1272/2008 and UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.

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 (39 mg/cm²) of the test item

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL of the vehicle

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution

Duration of treatment / exposure:

60 ± 1 minute

Duration of post-treatment incubation (if applicable):

approx. 42 hours

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

2.4

Vehicle controls validity:

not examined

Negative controls validity:

not valid

Positive controls validity:

not valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, no additional controls were necessary.
- Colour interference with MTT: mixture 25 mg of the test item per 300 µL aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, no additional controls were necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.773).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.4 %)
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.3 % - 1.8 %).
Please also refer to the field "An other information on results incl. tables" below.

Table 1: Result of the test item potassium neodecanoate

Name	Negative Control (NK)			Positive Control (PC)			Test material (TM)
Tissue	1	2	3	1	2	3	1	2	3
absolute OD570	1.736	1.682	1.746	0.090	0.100	0.101	0.080	0.088	0.078
	1.717	1.713	1.741	0.097	0.105	0.103	0.084	0.093	0.079
OD570(blank-corrected)	1.693	1.639	1.703	0.047	0.056	0.058	0.037	0.044	0.035
	1.674	1.670	1.698	0.054	0.062	0.060	0.041	0.049	0.036
mean OD570of the duplicates (blank-corrected)	1.684	1.654	1.701	0.050	0.059	0.059	0.039	0.047	0.036
total mean OD570of 3 replicate tissues (blank-corrected)	1.680*			0.056			0.040
SD OD570	0.023			0.005			0.006
relative tissue viability [%]	100.2	98.5	101.3	3.0	3.5	3.5	2.3	2.8	2.1
mean relative tissue viability [%]	100.0			3.3**			2.4
SD tissue viability [%]***	1.4			0.3			0.3
CV [% viabilities]	1.4			9.0			14.3

^* Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

^** Mean relative tissue viability of the three positive control tissues is ≤ 20%.

^*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%

Table 2: Historical data

	Mean OD570±30nmNK	Mean Relative Viability [%] PC	SD Viability [%]
Mean	1.843	4.3	4.2
SD	0.286	2.2	4.7
n	22	22	84

Historical data were generated from 2015 to 2017

Interpretation of results:

other: Category 1 (corrosive) or Category 2 (irritant) based on GHS criteria

Conclusions:

The test item, potassium neodecanoate, is either corrosive or irritating to the skin. Since the RhE test methods covered by OECD TG 439 cannot resolve between UN GHS Categories 1 or 2, further information on skin corrosion is required to decide on its final classification.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

2018-04-04

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:

2017-10-09

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry and cool

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Characteristics of donor animals: cattle was between 17 and 20 months old.
- Storage, temperature and transport conditions of ocular tissue: fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories
- Time interval prior to initiating testing: immediately after arrival of the eyes, cornea preparation was initiated and was used for BCOP testing on the same day.

Vehicle:

physiological saline

Remarks:

0.9 % NaCl

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL of the test item preparation
- Concentration (if solution): 10 % w/v concentration

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- eyes were examined for defects and any defective eyes were discarded. Eyes with scratches or any kind of opacity were not used.
- tissue surrounding the eyeball was pulled away and the cornea was excised.
- isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (BASF, Duratec) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with pre-warmed RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI).
- corneas were incubated for one hour at 32 ± 1 °C for equilibration in an air incubator.

QUALITY CHECK OF THE ISOLATED CORNEAS
- after the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI.
- an initial measurement was performed on each of the corneas using the opacitometer.
- three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas.
- the illuminance of each cornea was read and recorded.
- only corneas that had an initial illuminance reading I > I0/1.1651 lux (an equivalent to the opacity threshold of 7 as listed in OECD 437) were used for the assay.

APPLICATION DOSE AND EXPOSURE TIME
- medium was removed from the anterior chamber and replaced with the test item or control.
- 750 μL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
- after 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed.

REMOVAL OF TEST SUBSTANCE/CONTROL SUBSTANCES
- epithelium was washed at least three times with MEM (containing phenol red).
- once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).

METHODS FOR MEASURED ENDPOINTS:
- anterior chamber was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C.
- each cornea was observed visually and pertinent observations were recorded.
- corneas were visually examined for tissue peeling, residual test chemical and non-uniform opacity patterns and observation were recorded.
- after the illuminance measurement was performed, the medium was removed from both chambers of the holder.
- posterior chamber was refilled with fresh complete RPMI.
- 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C.
- then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Evaluation of the opacity:
- the following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:
Opacity = ((I0/I) - b)/ a
with a = 0.025 and b = 0.9894
- value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated annually. This I0 value is than calculated to the respective data of the opacitometer and the data according to guideline (opacity < 7). So the initial illuminance can be calculated and corneas below this value will be discarded.
- change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

Evaluation of the permeability:
- mean OD490 for the blank cuvettes was calculated.
- mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490).
- any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor.
- final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
- mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)
For the IVIS cut-off values for identifying test substances as inducing serious eye damage and test substances not including eye irritation or serious eye damage please refer to table 1 in the field "Any other information onmaterial and methods incl. tables" below.

ACCEPTABILITY CRITERIA
- the BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
- the negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Remarks:

(mean)

Value:

81.34

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All 3 corneas treated with potassium neodecanoate showed complete opacity of the tissue.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
- Acceptance criteria met for positive control: the in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The evaluation of acceptability criteria was performed by using the following historical data:
- for evaluation of the validity of the positive control, the historical mean IVIS score as obtained from 2015 until 2018 was used (please refer to the tables in the field "Any other information on results incl. tables").
- for the evaluation of the validity of the negative control, the historical upper limits of the opacity and permeability values as obtained in 2017 and 2018 were used (please refer to the tables in the field "Any other information on results incl. tables" below)

Please also refer for results to the field "Any other information on results incl. tables" below

Table 1: Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	1.11	1.61	0.50
2		1.11	1.87	0.75
3		1.11	2.16	1.05
MV		1.11	1.88	0.77
4	Positive Control	3.19	34.99	31.80	31.03
5		2.16	29.80	27.64	26.87
6		2.27	29.49	27.22	26.45
MV		2.54	31.43	28.89	28.12
7	Test Item	0.02	42.86	42.84	42.08
8		0.19	46.18	46.00	45.23
9		1.61	48.94	47.33	46.56
MV		0.61	46.00	45.39	44.62

MV = mean value

Table 2: Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative Control	0.022
2		0.045
3		0.015
MV		0.027
4	Positive Control	0.985	0.958
5		1.166	1.139
6		0.905	0.878
MV		1.019	0.991
7	Test Item	2.460	2.433
8		2.390	2.363
9		2.575	2.548
MV		2.475	2.448

MV = mean value

Table 3: In vitro irritation score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	0.50	0.022
2		0.75	0.045
3		1.05	0.015
MV		0.77	0.027	1.18
4	Positive Control	31.03	0.958
5		26.87	1.139
6		26.45	0.878
MV		28.12	0.991	42.99
7	Test Item	42.08	2.433
8		45.23	2.363
9		46.56	2.548
MV		44.62	2.448	81.34

MV = mean value

Table 4: Historical mean in vitro irritation score of the positive control from February 2015 until April 2018

	IVIS Positive Control – Ethanol 100%
Mean Value (MV)	48.57
Standard Deviation (SP)	9.69
MV-2xSD	29.20
MV+2xSD	67.94
Number of Replicates providing Historical Mean: 50

Positive controls are updated after every single experiment or at least every 3 months

Table 5: Historical data on opacity and permeability of the positive control (Ethanol 100 %) from August 2017 until April 2018

Number of Replicates Providing Historical Mean	Opacity		Permeability		IVIS
	Change of Opacity Value	Corrected Opacity Value	OD490 Value	Corrected OD490 Value
1	37.495	37.899	1.013	1.004	53.36
	27.335	27.739	1.147	1.138
	30.218	30.622	2.120	2.111
2	24.492	23.921	1.442	1.436	49.70
	31.754	31.182	1.108	1.102
	30.259	29.688	1.755	1.749
3	40.460	39.546	1.270	1.266	49.84
	28.925	28.010	0.949	0.945
	29.064	28.149	1.381	1.377
4	22.710	22.292	1.182	1.178	52.16
	35.986	35.567	1.780	1.776
	24.036	23.618	2.050	2.046
5	25.61	24.97	2.025	2.014	63.38
	28.89	28.25	2.920	2.909
	27.82	27.18	2.405	2.394
6	22.66	21.94	1.407	1.399	42.08
	22.03	21.31	1.354	1.346
	22.31	21.60	1.355	1.347
7	31.80	31.03	0.985	0.958	42.99
	27.64	26.87	1.166	1.139
	27.22	26.45	0.905	0.878
Mean Value (MV)	28.511	27.993	1.510	1.501	50.50
Standard Deviation (SD	5.022	5.109	0.534	0.536	7.13
MV- 2xSD	18.467	17.774	0.442	0.429	36.24
MV+2xSD	38.555	38.212	2.579	2.573	64.77

Table 6: Historical mean in vitro irritation score of the negative control from February 2015 until April 2018

	IVIS Negative Control – NaCl 0.9 %
Mean Value (MV)	0.81
Standard Deviation (SD)	0.64
MV-2xSD	-0.48
MV+2xSD	2.09
Number of Replicates providing Historical Mean: 50

Negative controls are updated every single experiment or at least every 3 months

Table 7: Historical data on opacity and permeability of the negative control (NaCl 0.9 %) from August 2017 until April 2018

Number of Replicates Providing Historical Mean	Opacity	Permeability
	Change of Opacity Value	OD490 Value	IVIS
1	-0.57	0.009	-0.27
	-0.11	0.013
	-0.53	0.004
2	-0.25	0.004	0.66
	0.80	0.005
	1.17	0.008
3	0.11	0.004	0.48
	0.68	0.005
	0.47	0.003
4	0.19	0.008	0.18
	-0.12	0.005
	0.00	0.018
5	0.11	0.005	0.81
	0.74	0.022
	1.06	0.007
6	0.26	0.011	0.83
	1.21	0.004
	0.68	0.008
7	0.50	0.022	1.18
	0.75	0.045
	1.05	0.015
Mean Value (MV)	0.39	0.01	0.55
Standard Deviation (SD	0.54	0.01	0.48
MV- 2xSD	-0.69	-0.01	-0.40
MV+2xSD	1.47	0.03	1.51

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

According to the bovine corneal opacity and permeability assay, since the IVIS was > 55 (IVIS score: 81.34), the test item Potassium neodecanoate can be considered as requiring classification for eye irritation or serious eye damage.
According to the Regulation (EC) No 1272/2008 and subsequent adaptions, the substance is classified for serious eye damage (Category 1; H318).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion/irritation

The substance was observed to be either corrosive or irritating to the skin in a reliable in vitro skin irritation/corrosion study according to OECD 439. OECD TG 439 cannot resolve between UN GHS Categories 1 or 2, thus further information on skin corrosion is required to decide on its final classification.

 

The substance was tested in an in vitro skin corrosion test according to OECD 435 and is not corrosive to the skin.

Justification for classification or non-classification

Skin corrosion/irritation

Potassium neodecanoate does possess a skin irritating potential based on in vitro OECD 439 and 435 tests and does require classification as skin irritant (Category 2, H315) according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation

The substance does possess an eye irritating potential based on an in vitro OECD 437 test and does require classification as serious eye damage (Category 1; H318) according to Regulation (EC) No 1272/2008 and its subsequent adaptations.