Registration Dossier

Administrative data

Description of key information

Skin corrosion/irritation: irritating (OECD 435 and OECD 439; GLP)

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

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-04-10
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
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: room temperature, in a tightly closed

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test item was crushed to a fine powder.
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:
The CORROSITEX™ Assay is a standardized, quantitative in vitro test for skin corrosivity and has been validated by the ECVAM for testing acids, bases and their derivatives (ECVAM, 2000)*. The bio-barrier membrane is constructed to have physico-chemical properties similar to rat skin.

Reference:
- ECVAM (2000) ESAC statement on the application of the Corrositex® assay for skin corrosivity testing
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, CA 92614)
- Components: a synthetic macromolecular bio-barrier and a chemical detection system (CDS)
- 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 (approx. 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
In order to test whether the test system is suitable for the test item, 100 mg of the test substance was added to the Qualify test tube. The vial was shaken to allow dissolution of the test substance and let stand for one minute. If the colour or consistency of the CDS changes at the sample/testing fluid interface, the test item is qualified for the assay. If no reaction is observed within five minutes, the sample is not qualified for the CORROSITEX TM 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 was added to the tubes labelled 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 items 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 (1995)*.

TEMPERATURE USED DURING TREATMENT: room temperature (17 - 25 °C)

METHOD OF DETECTION
- Chemical or electrochemical detection system: chemical detection system (CDS)

METHOD OF APPLICATION (CLASSIFICATION TEST):
The CDS vials were warmed to room temperature (17 - 25˚C) before using. Four vials were utilized for test item sample replicate testing. One vial was utilized for a positive control sample and another vial for a negative control. Lastly, one vial served as a CDS colour control. 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 was 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. 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.

INTERPRETATION OF THE RESULTS:
For Category 1 substances, test chemicals are categorized as non-corrosive in case no colour change occurs after 240 minutes. For Category 2 substances, test chemicals are categorized as non-corrosive in case no colour change occurs after 60 minutes. The start time difference for each vial was subtracted from the final time to determine the net response time.
The time (in minutes) elapsed between application and barrier penetration for the test substance was recorded in tabular form as individual replicate data.
The mean time (± standard deviation) of the four sample replicates to activate the CDS was calculated and reported in tabular form. Using the table as shown in the field "Any other information on materials and methods incl. tables" below, the test item was categorised.

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.
The exact breakthrough time of the positive control should be determined to demonstrate, that the response is in the acceptable historical range of breakthrough times for the positive control (mean ± 2 - 3 standard deviations).

*Reference:
- InVitro International (1995), Corrositex® Reference Manual
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg of the test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 500 µL of citric acid (10%) in aqua dest.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 500 µL of phosphoric acid (85 %)
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 examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Colour change or structural change was not observed up to 60 minutes (treatment period)
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.

CATEGORIZATION 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:
- Acceptance criteria met for negative control: negative control did not activate the CDS before 60 minutes (> 60 minutes).
- Acceptance criteria met for positive control: positive control activated the CDS between 3 - 60 minutes (21.08 min).

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

Table 1. Results of the Test Item Fatty acids, C9-13-neo-, potassium salts

 

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

21.08

yes

no

Negative control

> 60

no

no

Interpretation of results:
GHS criteria not met
Conclusions:
The test item 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.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-21 to 2018-03-23
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:
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: room temperature, in a tightly closed container
Test system:
human skin model
Source species:
human
Cell type:
other: normal, human 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 closely 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 number: 25888

TEST FOR DIRECT MTT REDUCTION
- to check the non-specific MTT-reducing capability of the test item 25 mg of the test item were 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 were 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).
- the mixture showed no colouring detected by unaided eye assessment. So the additional functional test with viable tissues and the quantitative corrections were not necessary.

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 at least for 2 hours with shaking on a plate shaker.
Before using the extracts, the plate had been 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 corresponded 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 concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance. 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
Firstly, 25 µL of the vehicle were applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm²) of the test item were applied directly atop the EpiDermTM tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue by using a bulb-headed Pasteur pipette.

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:
4.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
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 turned blue/purple. The coloured test material or MTT reducing substance was classified as “Irritant” i.e. mean tissue viability is < 50%. Therefore, no correction procedures were necessary.
- Colour interference with MTT: mixture 25 mg of the test item per 300 µL aqua dest. and/or per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, an additional test with viable tissues (without MTT addition) was not necessary (NSC equalled 0%).

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: 1.385).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (2.5 %)
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.5 % - 15.4 %).
- The absorbance values were not below historically established boundaries.

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

Table 1: Result of the test itemfatty acids, C9-13-neo-, potassium salts

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

1.156

1.505

1.496

0.057

0.092

0.070

0.109

0.098

0.110

1.136

1.498

1.521

0.060

0.100

0.081

0.111

0.099

0.114

Mean Absolute

OD570

1.385****

0.077

0.107

OD570(Blank

Corrected)

1.114

1.463

1.454

0.014

0.049

0.028

0.067

0.056

0.068

1.094

1.456

1.479

0.018

0.057

0.038

0.068

0.056

0.072

Mean OD570of

the Duplicates (Blank Corrected)

1.104

1.459

1.466

0.016

0.053

0.033

0.068

0.056

0.070

Total Mean OD570of 3

Replicate Tissues (Blank Corrected)

1.343*

0.034

0.064

SD of Mean

OD570 of the Duplicates (Blank Corrected)

0.207

0.019

0.007

Relative Tissue

Viability [%]

82.2

108.6

109.2

1.2

4.0

2.5

5.0

4.2

5.2

Mean Relative

Tissue Viability [%]

100.0

2.5**

4.8

SD of Relative

Tissue Viability [%]***

15.4

1.4

0.5

CV [% Viabilities]

15.4

54.4

11.4

* Blank-corrected mean OD570 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) of relative tissue viabilityobtained from the three concurrently tested tissues for test item, positive and negative control is  18%

**** Mean absolute OD570of the negative control tissues is 0.8 and 2.8.

Table 2: Historical data:

 

Mean Absolute OD570±30nmNK

MeanAbsoluteOD570±30nmPC

Mean Relative Viability [%] PC

SD Viability [%]
NK, PC, TI

Mean

1.861

0.114

3.7

4.4

SD

0.247

0.033

1.5

4.1

Range of
LCL – UCL

1.367 – 2.355

0.048 – 0.181

0.7 – 6.8

0.0 – 12.5

n

25

25

25

117

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,fatty acids, C9-13 neo, potassium salts, 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:
2018-03-28
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: room temperature, in a tightly closed container
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Characteristics of donor animals (e.g. age, sex, weight): age of the cattle was between 18 and 23 months
- 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
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
The test item was treated as a neat surfactant. The test item was suspended in 0.9 % NaCl to give a 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 (Duratec GmbH) 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 substance mixture or control substances were 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 more than three times with MEM (containing phenol red), since residual test material could not be removed.
- 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.
- 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 in horizontal position.
- 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 periodically. This I0 value was than calculated to the respective data of the opacitometer and the data according to guideline (opacity < 7). So the initial illuminance could be calculated and corneas below this value were discarded.
- change in opacity for each cornea (test item, positive and negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values of test item treated cornea or positive control were corrected by subtracting from each the average change in opacity observed for the negative-control corneas to obtain a corrected opacity. The mean corrected 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)
To determine the IVIS of the positive control and the test item, the corrected opacity and OD490 values were used.
For the IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) 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:
121.15
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
All 3 corneas treated with fatty acids, C9-13-neo-, potassium salts showed severe opacity of the tissue, with partially spotted test item residues.
Relative to the negative control, the test item caused a severe increase of corneal opacity and permeability in all 3 corneas.

The experiment showed residual of fatty acids, C9-13-neo-, potassium salts on the corneas. The results of the opacity measurements are therefore confounded by the presence test item material. Since the residual material could not be removed by non-invasive methods, such as additional rinsing, it is concluded that the in vitro eye irritancy potential of fatty acids, C9-13-neo-, potassium salts cannot be assessed in the bovine corneal opacity and permeability assay. Due to these technical limitations, the study cannot be performed with fatty acids, C9-13-neo-, potassium salts according to the guideline OECD437.

Acceptance of results:
- Acceptance criteria met for negative control: the negative control responses resulted in opacity and permeability values that are less than the respective established upper limits for background opacity and permeability.
- Acceptance criteria met for positive control: the IVIS of the positive control falls within two standard deviations of the current historical mean

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

2.20

2.46

0.26

 

2

2.73

3.94

1.21

 

3

2.27

2.96

0.68

 

MV

2.40

3.12

0.72

 

4

Positive
Control

3.54

26.20

22.66

21.94

5

3.98

26.01

22.03

21.31

6

3.70

26.01

22.31

21.60

MV

3.74

26.08

22.34

21.62

7

Test Item

1.58

40.19

38.62

37.90

8

2.20

49.61

47.42

46.70

9

0.22

45.25

45.03

44.31

MV

1.33

45.02

43.69

42.97

MV = mean value

Table 2: Permeability

Cornea
No.

Test Item

OD490

Corrected
OD490 Value

1

Negative
Control

0.011

 

2

0.004

 

3

0.008

 

MV

0.008

 

4

Positive
Control

1.407

1.399

5

1.354

1.346

6

1.355

1.347

MV

1.372

1.364

7

Test Item

5.470

5.462

8

4.430

4.422

9

5.760

5.752

MV

5.220

5.212

MV = mean value

Table 3: In vitro irritation score

Cornea
No.

Test Item

Corrected
Opacity

Value

Corrected
OD490

Value

IVIS

1

Negative
Control

0.26

0.011

 

2

1.21

0.004

 

3

0.68

0.008

 

MV

0.72

0.008

0.83

4

Positive
Control

21.94

1.399

 

5

21.31

1.346

 

6

21.60

1.347

 

MV

21.62

1.364

42.08

7

Test Item

37.90

5.462

 

8

46.70

4.422

 

9

44.31

5.752

 

MV

42.97

5.212

121.15

MV = mean value

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

 

IVIS
Positive Control - Ethanol

100 %

Mean Value (MV)

48.69

Standard Deviation (SD)

9.75

MV- 2xSD

29.18

MV+2xSD

68.19

Number of Replicates providing Historical Mean: 49

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 March 2018

Number

of Replicates Providing Historical Mean

Cornea No.

Opacity

Permeability

IVIS

Change of
Opacity Value

Corrected
Opacity

Value

OD490 Value

Corrected
OD490

Value

2017

1

4

37.495

37.899

1.013

1.004

53.36

 

5

27.335

27.739

1.147

1.138

 

6

30.218

30.622

2.120

2.111

 

2

4

24.492

23.921

1.442

1.436

49.70

 

5

31.754

31.182

1.108

1.102

 

6

30.259

29.688

1.755

1.749

3

4

40.460

39.546

1.270

1.266

49.84

5

28.925

28.010

0.949

0.945

6

29.064

28.149

1.381

1.377

 

4

4

22.710

22.292

1.182

1.178

52.16

 

5

35.986

35.567

1.780

1.776

 

6

24.036

23.618

2.050

2.046

2018

6

4

25.61

24.97

2.025

2.014

63.38

 

5

28.89

28.25

2.920

2.909

 

6

27.82

27.18

2.405

2.394

 

7

4

22.66

21.94

1.407

1.399

42.08

 

5

22.03

21.31

1.354

1.346

 

6

22.31

21.60

1.355

1.347

Mean

Value (MV)

28.448

27.972

1.592

1.586

51.75

Standard

Deviation (SD)

5.375

5.473

0.533

0.532

6.92

MV- 2xSD

17.699

17.025

0.526

0.522

37.91

MV+2xSD

39.197

38.918

2.658

2.649

65.59

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

 

IVIS Negative Control - NaCl 0.9 %

Mean Value (MV)

0.80

Standard Deviation (SD)

0.65

MV- 2xSD

-0.49

MV+2xSD

2.10

Number of Replicates providing Historical Mean: 49

Negative controls are updated after 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 March 2018

Number of

Replicates Providing Historical Mean

Cornae No.

Opacity

Permeability

IVIS

Change of
Opacity Value

OD490 Value

2017

1

1

-0.57

0.009

-0.27

2

-0.11

0.013

3

-0.53

0.004

2

1

-0.25

0.004

0.66

2

0.80

0.005

3

1.17

0.008

3

1

0.11

0.004

0.48

2

0.68

0.005

3

0.47

0.003

2018

4

1

0.19

0.008

0.18

2

-0.12

0.005

3

0.00

0.018

 

1

0.11

0.005

 

5

2

0.74

0.022

0.81

 

3

1.06

0.007

 

6

1

0.26

0.011

 

2

1.21

0.004

0.83

3

0.68

0.008

 

Mean Value (MV)

0.15

0.01

0.37

Standard Deviation (SD)

0.54

0.00

0.43

MV- 2xSD

-0.92

0.00

-0.49

MV+2xSD

1.22

0.02

1.23

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The in vitro eye irritancy potential of fatty acids, C9-13-neo-, potassium salts cannot be assessed in the bovine corneal opacity and permeability assay due to residual test item on the cornea, confounding the transmission measurements. However, based on the qualitative visual appraisal of the cornea being markedly opaque, a conservative classification of fatty acids, C9-13-neo-, potassium salts being severely damaging to eye (H318) appears justified, In conclusion, Fatty acids, C9-13-neo-, potassium salts needs to be classified as serious eye damaging (EU CLP/ UN GHS Category 1).
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. As the results of an OECD 439 test are not suitable to differentiate between skin categories 1 and 2, the substance was tested in an in vitro skin corrosion test.

Fatty acids, C9 -13 neo, potassium salts was tested in an in vitro skin corrosion test according to OECD 435 and the substance was not observed to be corrosive to the skin.

Eye irritation:

The substance was observed to be corrosive/severe irritanting to the eyes in an in vitro eye irritation study according to OECD 437.

Justification for classification or non-classification

Skin irritation:

Fatty acids, C9 -13 neo, potassium salts does possess a skin irritation potential based on in vitro OECD 435 and 439 studies. The substance does require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations (Category 2; H315).

Eye irritation:

The in vitro eye irritancy potential of fatty acids, C9-13-neo-, potassium salts cannot be assessed in the bovine corneal opacity and permeability assay due to residual test item on the cornea, confounding the transmission measurements. However, based on the qualitative visual appraisal of the cornea being markedly opaque, a conservative classification of fatty acids, C9-13-neo-, potassium salts being severely damaging to eye (H318) appears justified, In conclusion, Fatty acids, C9-13-neo-, potassium salts needs to be classified as serious eye damaging (EU CLP/ UN GHS Category 1).