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EC number: 248-375-1 | CAS number: 27253-33-4
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Description of key information
Skin corrosion: not corrosive (OECD 435; GLP)
Skin irritation: irritating (OECD 439; GLP)
Eye irritation: causes serious eye irritation (OECD 405; GLP)
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-08-02 to 2017-08-25
- 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: tightly closed container at room temperature - 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: 25834 & 25838
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 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%. 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:
- 28.7
- Vehicle controls validity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- NOTE: in the first experiment the mean relative tissue viability (% negative control) was ≥ 50% (63.3%) after 60 minute treatment and 42 hour post-incubation. The single values of the three replicates treated identically were on both sides of the classification cut-off (66.6%, 81.4% and 41.8%). Standard deviation of the three test item treated tissues exceeded the 18% threshold. Therefore, the experiment was considered invalid and repeated. For the sake of completeness, the results of the first experiment are included in the field "Any other information on results incl. tables" below.
- 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 (second experiment only):
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.752).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (4.4 %)
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability of replicate tissues of all dose groups was ≤ 18 % (0.1 % - 7.8 %).
Please also refer to the field "An other information on results incl. tables" below. - Interpretation of results:
- other: Category 1 (corrosive) or Category 2 (irritant) based on GHS criteria
- Conclusions:
- The test item, calcium 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:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-11-21 to 2017-11-21
- 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:
- 1999-06
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICCVAM recommended performance standards for in vitro test methods for skin corrosion. NIEHS, NIH publication No. 04-4510
- Version / remarks:
- 2004-05
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICCVAM: Corrositex- A validated and accepted dermal corrosion test method for classifying substances according to the UN packing groups.
- Version / remarks:
- 2003-10
- 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 - 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 positive control
- 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
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 as the mean time, required to activate the CDS, was > 60 min. (category 2).
- 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.) - Interpretation of results:
- GHS criteria not met
- Conclusions:
- Calcium 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.
Referenceopen allclose all
Table 1: Result of the test item calcium neodecanoate (Experiment 1)
Name |
Negative Control (NK) |
Positive Control (PC) |
Test material (TM) |
||||||
Tissue |
1 |
2 |
3 |
1 |
2 |
3 |
1 |
2 |
3 |
absolute OD570 |
1.755 |
1.853 |
1.950 |
0.112 |
0.104 |
0.087 |
1.210 |
1.497 |
0.758 |
1.819 |
1.847 |
1.671 |
0.120 |
0.106 |
0.092 |
1.238 |
1.476 |
0.811 |
|
OD570(blank-corrected) |
1.712 |
1.811 |
1.907 |
0.069 |
0.061 |
0.044 |
1.167 |
1.454 |
0.716 |
1.776 |
1.804 |
1.628 |
0.078 |
0.063 |
0.049 |
1.195 |
1.433 |
0.768 |
|
mean OD570of the duplicates (blank-corrected) |
1.744 |
1.808 |
1.767 |
0.073 |
0.062 |
0.047 |
1.181 |
1.443 |
0.742 |
total mean OD570of 3 replicate tissues (blank-corrected) |
1.773* |
0.061 |
1.122 |
||||||
SD OD570 |
0.032 |
0.013 |
0.355 |
||||||
relative tissue viability [%] |
98.4 |
101.9 |
99.7 |
4.1 |
3.5 |
2.6 |
66.6 |
81.4 |
41.8 |
mean relative tissue viability [%] |
100.0 |
3.4** |
63.3 |
||||||
SD tissue viability [%]*** |
1.8 |
0.8 |
20.0 |
||||||
CV [% viabilities] |
1.8 |
22.1 |
31.6 |
* Blank-corrected mean OD570 nmof 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%. Criterion failed for test item treated tissues
Table 2: Result of the test item calcium neodecanoate (Experiment 2)
Name |
Negative Control (NK) |
Positive Control (PC) |
Test material (TM) |
||||||
Tissue |
1 |
2 |
3 |
1 |
2 |
3 |
1 |
2 |
3 |
absolute OD570 |
1.844 |
1.804 |
1.764 |
0.117 |
0.118 |
0.117 |
0.521 |
0.426 |
0.692 |
1.829 |
1.767 |
1.763 |
0.118 |
0.121 |
0.125 |
0.535 |
0.413 |
0.692 |
|
OD570(blank-corrected) |
1.801 |
1.761 |
1.721 |
0.074 |
0.075 |
0.074 |
0.478 |
0.383 |
0.649 |
1.786 |
1.724 |
1.720 |
0.075 |
0.078 |
0.082 |
0.492 |
0.370 |
0.649 |
|
mean OD570of the duplicates (blank-corrected) |
1.794 |
1.743 |
1.721 |
0.075 |
0.077 |
0.078 |
0.485 |
0.377 |
0.649 |
total mean OD570of 3 replicate tissues (blank-corrected) |
1.752* |
0.076 |
0.504 |
||||||
SD OD570 |
0.037 |
0.002 |
0.137 |
||||||
relative tissue viability [%] |
102.4 |
99.4 |
98.2 |
4.3 |
4.4 |
4.5 |
27.7 |
21.5 |
37.1 |
mean relative tissue viability [%] |
100.0 |
4.4** |
28.7 |
||||||
SD tissue viability [%]*** |
2.1 |
0.1 |
7.8 |
||||||
CV [% viabilities] |
2.1 |
2.3 |
27.3 |
* Blank-corrected mean OD570 nmof 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 3: Historical data
|
Mean OD570±30nm Negative control (NK) |
Mean Relative Viability [%] Positive control (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
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 |
19.90 |
Yes |
No |
Negative control |
> 60 |
No |
No |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018-01-01 to 2018-01-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 405 (Acute Eye Irritation / Corrosion)
- Version / remarks:
- 2017-10-09
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- signed on 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 - Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or tissues and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 15 weeks old
- Weight at study initiation: 3.7 kg
- Housing: individually housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm²
- Diet (ad libitum): autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Water (ad libitum): tap water
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3 °C
- Relative humidity: 55 ± 10%
- Air changes: at least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12 - Vehicle:
- unchanged (no vehicle)
- Controls:
- no
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g of the test item applied in the conjunctival sac of one eye. Untreated eye served as control. - Duration of treatment / exposure:
- not applicable
- Observation period (in vivo):
- 1, 24, 48 and 72 hours as well as 4 to 21 days after test item application
- Duration of post- treatment incubation (in vitro):
- not applicable
- Number of animals or in vitro replicates:
- 1 male rabbit
- Details on study design:
- PREPARATION OF THE ANIMALS
Within 24 hours before the test and immediately prior to the application both eyes of the animal were examined.
Approx. 22 hours before the application the eyes were examined with the aid of a fluorescein solution (Fluoreszein SE Thilo®). The eyes were rinsed with physiological saline 0.9 % NaCl after the examination. The animal did not showed eye irritation, ocular defects, or pre-existing corneal injury.
INITIAL AND CONFIRMATORY TEST
The in vivo test was performed initially using one animal. The results of the initial test indicated that the test item is corrosive or severe irritant to the eye using the procedure described. Therefore, no additional animals were treated due to animal welfare reasons.
USE OF TOPICAL ANESTHETICS AND SYSTEMIC ANALGESICS
One hour before the application of the test item, 0.01 mg/kg of buprenorphine (Temgesic® 0.3 mg/mL) was administered subcutaneously in order to achieve a therapeutic level of systemic analgesia. Approx. 5 minutes prior to the application of the test item, 1-2 drops of an ocular anaesthetic (Proparakaine-POS® hydrochloride ophthalmic 0.5% solution) were administered in both the treated and the control eye of the animal.
To prevent pain and distress after the application of the test item the animal was treated with doses of buprenorphine and meloxicam (Metacam® 5 mg/mL) to provide a continued therapeutic level of systemic analgesia. Treatment with the analgesic medication was conducted from 11 hours post-application (day 0) upto 6 days post-application.
REMOVAL OF TEST SUBSTANCE
- Washing: treated eye was rinsed with physiological saline 0.9 % NaCl.
- Time after start of exposure: 1 hour after the application
The eyes were not rinsed to remove residues of the test item, foreign bodies or incrustation 24 hours after application.
SCORING SYSTEM: according to Draize scale
TOOL USED TO ASSESS SCORE: slit lamp biomicroscope / fluorescein
24 hours post-application and from then on daily until end of the observation period, the treated eye was examined with the aid of a fluorescein solution and a slit lamp biomicroscope. After the examination the eye was rinsed with physiological saline 0.9% NaCl.
OBSERVATIONS
- body weight: prior to the administration and at least at the end of the observation period
- clinical observations: nature, severity and duration were recorded - Irritation parameter:
- cornea opacity score
- Basis:
- mean
- Remarks:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 1.67
- Max. score:
- 4
- Reversibility:
- not reversible
- Irritation parameter:
- iris score
- Basis:
- mean
- Remarks:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 1
- Max. score:
- 2
- Reversibility:
- fully reversible within: 10 days
- Irritation parameter:
- conjunctivae score
- Basis:
- mean
- Remarks:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 2.33
- Max. score:
- 3
- Reversibility:
- not reversible
- Irritation parameter:
- chemosis score
- Basis:
- mean
- Remarks:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 1.67
- Max. score:
- 4
- Reversibility:
- not reversible
- Irritant / corrosive response data:
- Animal # 1:
- conjunctival redness: the animal mainly shows slight to moderate conjunctival redness (grade 1 or 2) from 1 hour until day 21 post-application. The effect was not fully reversible within the observation period of 21 days after application of the test material.
- conjuctival chemosis: moderate conjunctival chemosis (grade 2) was detected 1 hour post-application until 48 hours post-application and slight conjunctival chemosis (grade 1) was observed from 72 hours until day 21 post-application, except for day 8 post-application (no chemosis; grade 0). The effect was not fully reversible within the observation period of 21 days after application of the test material.
- iris: slight iris lesions (grade 1) was observed from 24 hours post-application until day 9 post-application. The effect was fully reversible within day 10 after application of the test material.
- corneal opacitiy: slight corneal opacity (grade 1) was observed throughout the whole observation period of 21 days, except for 24 hours and 48 hours after application (moderate corneal opacitiy ; grade 2). The effect was not reversible within 21 days.
Local effects were observed. The observed local effects mainly comprise moderate to slight hypersecretion, vascularisation from dorsal rim to corneal lesion, and slight unevenness on corneal surface.
Upon fluorescein examinations starting 24 hours post-application, the treated eye of the animal showed corneal lesions (starting with approx. 80% of the area) which were not completely reversible within 21 days. - Other effects:
- - clinical observations: neither mortality nor significant clinical signs of toxicity were observed.
- body weight: the body weight development was within the expected range. - Interpretation of results:
- Category 1 (irreversible effects on the eye) based on GHS criteria
- Conclusions:
- The substance is serious eye damaging.
According to the EC Regulation No. 1272/2008 and subsequent adaptations, the substance is classified as a serious eye damaging (Category 1; H318). - Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-08-09
- 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:
- 2013-07-26
- 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: cattle was between 16 and 60 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
- 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 substance mixture
- 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 substance mixture was applied directly onto the cornea by removing the window-locking ring and glass window prior to treatment.
- 750 µL of 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 were 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:
- 45.91
- Vehicle controls validity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- All 3 corneas treated with calcium neodecanoate showed severe 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 2017 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 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 - Interpretation of results:
- study cannot be used for classification
- Conclusions:
- According to the bovine corneal opacity and permeability assay, since the IVIS was between 3 and 55, no prediction can be made regarding the test item Calcium neodecanoate.
Referenceopen allclose all
Table 1: Opacity
Cornea No. |
Test Item |
Initial Opacity |
Final Opacity |
Change of Opacity Value |
Corrected Opacity Value |
1 |
Negative Control |
1.65 |
1.08 |
-0.57 |
|
2 |
1.65 |
1.54 |
-0.11 |
|
|
3 |
1.58 |
1.04 |
-0.53 |
|
|
MV |
1.63 |
1.22 |
-0.40 |
|
|
4 |
Positive Control |
3.11 |
40.60 |
37.49 |
37.90 |
5 |
2.88 |
30.21 |
27.34 |
27.74 |
|
6 |
3.03 |
33.25 |
30.22 |
30.62 |
|
MV |
3.01 |
34.69 |
31.68 |
32.09 |
|
7 |
Test Item |
1.76 |
34.52 |
32.76 |
33.16 |
8 |
2.57 |
35.71 |
33.13 |
33.54 |
|
9 |
1.11 |
45.25 |
44.14 |
44.54 |
|
MV |
1.82 |
38.49 |
36.68 |
37.08 |
MV = mean value
Table 2: Permeability
Cornea No. |
Test Item |
OD490 |
Corrected OD490 Value |
1 |
Negative Control |
0.009 |
|
2 |
0.013 |
|
|
3 |
0.004 |
|
|
MV |
0.009 |
|
|
4 |
Positive Control |
1.013 |
1.004 |
5 |
1.147 |
1.138 |
|
6 |
2.120 |
2.111 |
|
MV |
1.427 |
1.418 |
|
7 |
Test Item |
0.729 |
0.720 |
8 |
0.567 |
0.558 |
|
9 |
0.495 |
0.486 |
|
MV |
0.597 |
0.588 |
MV = mean value
Table 3: In vitro irritation score
Cornea No. |
Test Item |
Corrected Opacity |
Corrected OD490 Value |
IVIS |
1 |
Negative Control |
-0.57 |
0.009 |
|
2 |
-0.11 |
0.013 |
|
|
3 |
-0.53 |
0.004 |
|
|
MV |
-0.40 |
0.009 |
-0.27 |
|
4 |
Positive Control |
37.90 |
1.004 |
|
5 |
27.74 |
1.138 |
|
|
6 |
30.62 |
2.111 |
|
|
MV |
32.09 |
1.418 |
53.36 |
|
7 |
Test Item |
33.16 |
0.720 |
|
8 |
33.54 |
0.558 |
|
|
9 |
44.54 |
0.486 |
|
|
MV |
37.08 |
0.588 |
45.91 |
MV = mean value
Table 4: Historical mean in vitro irritation score of the positive control from February 2015 until August 2017
|
IVIS Positive Control – Ethanol 100% |
Mean Value (MV) |
48.38 |
Standard Deviation (SD) |
9.98 |
MV-2xSD |
28.41 |
MV+2xSD |
68.34 |
Number of Replicates providing Historical Mean: 44 |
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 October 2017
Incubation: 10 min |
||||||
Number of Replicates providing Historical Mean |
Cornea No. |
Opacity |
Permeability |
IVIS |
||
Change of Opacity Value |
Corrected Opacity Value |
OD490 Value |
Corrected OD490 Value |
|||
1 |
4 |
37.495 |
37.899 |
1.013 |
1.004 |
|
|
5 |
27.335 |
27.739 |
1.147 |
1.138 |
53.36 |
|
6 |
30.218 |
30.622 |
2.120 |
2.111 |
|
2 |
4 |
24.492 |
23.921 |
1.442 |
1.436 |
|
|
5 |
31.754 |
31.182 |
1.108 |
1.102 |
49.70 |
|
6 |
30.259 |
29.688 |
1.755 |
1.749 |
|
Mean Value (MV) |
30.259 |
30.175 |
1.431 |
1.424 |
51.530 |
|
Standard Deviation (SD) |
4.391 |
4.608 |
0.433 |
0.434 |
2.588 |
|
MV-2xSD |
21.477 |
20.960 |
0.564 |
0.557 |
46.354 |
|
MV+2xSD |
39.040 |
39.391 |
2.298 |
2.291 |
56.706 |
Table 6: Historical mean in vitro irritation score of the negative control from February 2015 until September 2017
|
IVIS Negative Control – NaCl 0.9 % |
Mean Value (MV) |
0.81 |
Standard Deviation (SD) |
0.68 |
MV-2xSD |
-0.56 |
MV+2xSD |
2.17 |
Number of Replicates providing Historical Mean: 44 |
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 October 2017
Number of Replicates providing Historical Mean |
Cornea No. |
Opacity |
Permeability |
IVIS |
Change of Opacity Value |
OD490 Value |
|||
1 |
1 |
0.234 |
0.008 |
1.49 |
|
2 |
1.738 |
0.008 |
|
|
3 |
0.800 |
0.098 |
|
2 |
1 |
0.978 |
0.019 |
2.52 |
|
2 |
3.920 |
0.022 |
|
|
3 |
1.617 |
0.028 |
|
3 |
1 |
-0.149 |
0.009 |
-0.50 |
|
2 |
-0.415 |
0.015 |
|
|
3 |
-1.427 |
0.009 |
|
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 |
|
Mean Value (MV) |
0.520 |
0.017 |
0.780 |
|
Standard Deviation (SD) |
1.296 |
0.023 |
1.254 |
|
MV-2xSD |
-2.072 |
-0.029 |
-1.727 |
|
MV+2xSD |
3.112 |
0.064 |
3.287 |
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.
Eye irritation
According to the bovine corneal opacity and permeability assay, since the IVIS was between 3 and 55,no prediction can be maderegarding the test itemCalcium neodecanoate.
Justification for classification or non-classification
Skin corrosion/irritation
Calcium neodecanoate does possess a skin irritation potential based on in vitro OECD 439 and OECD 435 tests and does require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations (Category 2; H315).
Eye irritation
Calcium neodecanoate does possess a serious eye damaging potential based on an in vivo OECD 405 test and does require classification as serious damaging to the eyes according to Regulation (EC) No 1272/2008 and its subsequent adaptations (Category 1; H318).
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