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EC number: 269-084-6 | CAS number: 68187-29-1
- 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
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 08 December 2016 to 09 December 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
- Version / remarks:
- 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EU Method B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- L-Glutamic acid, N-coco acyl derivs., compds. with triethanolamine (1:1)
- EC Number:
- 269-084-6
- EC Name:
- L-Glutamic acid, N-coco acyl derivs., compds. with triethanolamine (1:1)
- Cas Number:
- 68187-29-1
- Molecular formula:
- Not specified (UVCB substance)
- IUPAC Name:
- bis(tris(2-hydroxyethyl)azanium) (2S)-4-carboxy-2-dodecanamidobutanoate (2S)-4-carboxy-2-tetradecanamidobutanoate
- Test material form:
- solid: particulate/powder
- Details on test material:
- -Appearance: White to pale yellow solid
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Justification for test system used:
- The EPISKIN™(SM) model has been validated for corrosivity testing in an international trial and its use is recommended by the relevant OECD
guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study. - Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™(SM) (Manufacturer: SkinEthic, France)
- Tissue batch number(s): 16-EKIN-049
- Expiry date: 12 December 2016
- Use of this model is recommended by OECD 431.
- Quality control: All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories.
-Storage: The kits were found to be in good order at reception. They were kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8 °C until the initiation of the test.
INDICATOR FOR POTENTIAL FALSE VIABILITY
-Check for direct MTT reduction with the test material: Approximately 20 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37 °C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere for 3 hours and then any colour change was observed: Test materials which do not react with MTT turn yellow, test materials reacting with MTT turn blue or purple.
After three hours incubation, yellow colour of the mixture was detected; therefore additional controls were not used in the experiment.
- Check-method to detect the colouring potential of test material: Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol (simulating a tissue humid environment). As the test material had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non specific OD evaluation. These tissues followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.
MAIN STUDY
PRE-INCUBATION (DAY [-1])
- The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37 °C in an incubator with 5 % CO2 in a > 95 % humidified atmosphere.
APPLICATION (DAY 0)
-The Assay Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
- 20 mg of test material was applied evenly to the epidermal surface of each of two test material treated units and each additional control skin units and then 100 μL physiological saline was added to the test material to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.
-The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (22.9-24.2°C) covered with the plate lids.
REMOVAL OF TEST MATERIAL AND CONTROLS
- After the incubation time (4 hours), all test material treated tissues or also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly.
The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).
MTT TEST AND FORMAZAN EXTRACTION (DAY 0)
-MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two living colour control units). The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours (±15 minutes), protected from light.
-At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
-The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
- A blank sample containing 2 mL of acidified isopropanol was processed in parallel.
NUMBER OF REPLICATE TISSUES: 2
CELL VIABILITY MEASUREMENTS (DAY 1)
-Following the formazan extraction, 2 × 200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
-The instrument was verified and calibrated.
CALCULATIONS
The data calculation using two replicates is shown below. Results were calculated in a similar way when more replicates are used.
-Blank: The mean of the 6 blank OD values was calculated.
-Negative control: Individual negative control OD values (NCraw) were corrected with the mean blank OD:
OD Negative Control (ODNC) = ODNCraw – ODblank mean
The corrected mean OD of the 2 negative control values was also calculated: this corresponds to 100 % viability.
- Positive control: Individual positive control OD values (PCraw) were corrected with the mean blank OD:
OD Positive Control (ODPC) = ODPCraw – ODblank mean
The corrected mean OD of the 2 positive control values was calculated.
The % viability for each positive control replicate was calculated relative to the mean negative control:
Positive Control1 % = (ODPC1 / mean ODNC) ×100
Positive Control2 % = (ODPC2 / mean ODNC) ×100
– The mean value of the 2 individual viability % for positive control was calculated:
Mean PC % = (PC1 % + PC2 % ) / 2
Test material: Individual test material OD values (TTraw) were corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
The corrected mean OD of the 2 test material values was calculated. The % viability for each test material replicate was calculated relative to the mean negative control:
Treated Tissue1 % = (ODTT1 / mean ODNC) ×100
Treated Tissue2 % = (ODTT2 / mean ODNC) ×100
– The mean value of the 2 individual viability % for test material was calculated:
Mean TT % = (TT1 % + TT2 %) / 2
– The variability for 2 disks was calculated as:
(Disk1-Disk2)/((Disk1+Disk2)/2) x 100 %
-Data calculation for test materials having MTT-interacting potential:
Test materials that interfere with MTT can produce non specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value is corrected by the result of the additional controls before calculation of viability% as follows:
Non specific MTT reduction calculation (NSMTT%): NSMTT% = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD
ODKT: test material treated killed tissues OD
ODNC: negative control OD
If NSMTT% is ≤ 50 %, then true MTT metabolic conversion (TODTT) has to be undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test material treated viable tissues
– The % relative viability (RV%) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test material is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2
If NSMTT% is > 50% relative to the negative control: additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.
- Data calculation for test materials having colouring potential:
For test materials detected as able to stain the tissues the non specific OD was evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability % as detailed below:
Non Specific Colour % (NSCliving %): NSCliving % = (mean ODCTV / mean ODNC)×100
ODCTV: test material treated viable tissue (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
If NSC living % is ≤ 5 % then the normal calculation mode was used.
If NSC living % is > 5 % a nd ≤ 5 0 %, then additional correction (TODTT) has to be undertaken as follows:
TODTT = [ODTV - ODCTV]
ODTT: test material treated viable tissue (incubated with MTT)
ODCTV: test material treated viable tissue (not incubated with MTT)
The % relative viability (RV% %) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
The mean value of the 2 individual relative viability % for test material is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2
If NSC living % is > 50 % relative to the negative control, additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.
- Data calculation for test materials having both MTT-interacting and colouring potential:
For test materials detected as able to both stain the tissues and interfere with MTT may also require a third set of controls before calculation of the “true” viability %.
Non Specific Colour % with killed tissues (NSCkilled %):
NSCkilled % = (mean ODCTK / mean ODNC)×100
ODCTK: test material treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV +mean ODCTK]
ODTT: test material treated viable tissues (incubated with MTT)
ODKT: test material treated killed tissues OD
ODKNC: negative control killed tissues OD
ODCTV: test material treated viable tissues (not incubated with MTT)
ODCTK: test material treated killed tissues (not incubated with MTT)
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
The mean value of the 2 individual relative viability % for test material is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2
INTERPRETATION OF THE TEST RESULTS
For 2 disks:
-If both disks have mean viability of ≥ 35 % = Non Corrosive
-If both disks have mean viability of < 35 % = Corrosive (at the corresponding incubation period)
Otherwise:
-If the mean value is ≥ 35 % and the variability is less than 50 % = Non Corrosive
-If the mean value is < 35 % and the variability is less than 50 % = Corrosive
Otherwise:
If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35 %. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.
- Sub Category 1A: If corrosive after 3 min exposure
- Sub Category 1B: If not corrosive after 3 min exposure and corrosive after 1 hour exposure
- Sub Category 1C: If not corrosive after 1 hour exposure and corrosive after 4 hours exposure
-Non corrosive: If not corrosive after 4 hours exposure. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied: 20 mg with 100 µL physiological saline to ensure good contact with the epidermis
NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 0.9 % (w/v)
POSITIVE CONTROL
- Amount(s) applied: 50 µL - Duration of treatment / exposure:
- 4 hours (±10 min)
- Duration of post-treatment incubation (if applicable):
- 3 hours (±15 minutes) with MTT
- Number of replicates:
- The test was performed in duplicate.
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Mean
- Value:
- 86.7
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- ADDITIONAL CONTROLS
As the test material was coloured, two additional test material-treated tissues were used for the non specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.011, Non Specific Colour % (NSCliving %) was calculated as 1.3 %. This is below the threshold of 5 %, therefore correction due to colouring potential was not necessary.
VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1. The mean OD value for the test material treated skin samples showed 86.7 % relative viability.
VALIDITY OF THE TEST
-After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
-The mean OD value of the two negative control tissues was in the recommended range (0.836).
-The two positive control treated tissues showed 0.8 % viability demonstrating the proper performance of the assay.
- The difference of viability between the two test material-treated tissue samples in the MTT assay was 16.7 %.
- The difference of viability between the two negative control tissue samples in the MTT assay was 15.0 %.
-The mean OD value of the blank samples (acidified isopropanol) was 0.046.
-All these parameters were within acceptable limits and therefore the study was considered to be valid.
Any other information on results incl. tables
Table 1: Optical Density (OD) and the calculated relative viability % of the samples
Substance |
Run |
Optical density |
Viability (% RV) |
|
Measured |
Blank corrected |
|||
Negative control |
1 |
0.819 |
0.773 |
92.5 |
2 |
0.944 |
0.898 |
107.5 |
|
Mean |
- |
0.836 |
100.0 |
|
Positive control |
1 |
0.052 |
0.006 |
0.8 |
2 |
0.053 |
0.007 |
0.8 |
|
Mean |
- |
0.007 |
0.8 |
|
Test material |
1 |
0.710 |
0.664 |
79.5 |
2 |
0.831 |
0.785 |
94.0 |
|
Mean |
- |
0.725 |
86.7 |
Mean blank value was 0.046.
Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).
Applicant's summary and conclusion
- Interpretation of results:
- other: Not corrosive in accordance with EU criteria
- Conclusions:
- Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.
- Executive summary:
The potential of the test material to cause skin corrosion was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40 bis under GLP conditions using a human skin model assay.
Disks of EPISKIN™ (SM) were treated with the test material in duplicate and incubated for 4 hours at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.
Physiological saline (0.9 % (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, also performed in duplicate. Two additional disks were used to provide an estimate of colour contribution (NSCliving %) from the test material. For each treated tissue, viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35 % of the negative control, the test material is considered to be corrosive to skin.
Following exposure with the test material, the mean cell viability was 86.7 % compared to the negative control. This is above the threshold of 35 %, therefore the test material was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.
Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.
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