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EC number: 256-425-9 | CAS number: 49673-81-6
- 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
Eye irritation
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From April 17 to 19, 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
- Principles of method if other than guideline:
- Evaluation of eye irritation on 3D human corneal epithelium in vitro reconstituted (SkinEthic HCE) through the investigation of cytotoxicity by MTT test.
The test method is comparable to OECD guideline 492 (2017). - GLP compliance:
- no
- Remarks:
- the available study was originally run not for REACH purposes; however, it is scientifically valid and well documented.
Test material
- Reference substance name:
- L-lysine, S-carboxymethyl-L-cysteine salt
- IUPAC Name:
- L-lysine, S-carboxymethyl-L-cysteine salt
- Test material form:
- solid: particulate/powder
Constituent 1
Test animals / tissue source
- Details on test animals or tissues and environmental conditions:
- - Justification of the test method: evaluation of cell survival after the exposure to the substance through MTT assay. MTT assay method is a colorimetric assay that allows to determinate the percentage of living cells within a cell culture. This assay is based on the ability the mitochondrial succinate dehydrogenase enzyme to metabolise the tetrazolium salt, giving a colored compound.
- Description of the cell system used: human corneal epithelium in vitro reconstituted. A reconstructed artificial human skin model comprising transformed human corneal epitheliaI cells, kept for 5 days in chemically defined medium on inert polycarbonate filters. It was purchased from SkinEthic.
Test system
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- 30 mg
- Duration of treatment / exposure:
- 1 hour at room T.
- Duration of post- treatment incubation (in vitro):
- 16 hours at 37 °C, 5 % CO2.
- Number of animals or in vitro replicates:
- 3 replicates
- Details on study design:
- MTT assay
At the end of the exposure the MTT assay was performed to evaluate the cell survival on the skin units.
The MTT assay is simple, accurate and yields reproducible results. The key component is (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) or MTT (Sigma M-2128). This product is of yellowish colour in solution. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, leading to the formation of purple crystals which are insoluble in aqueous solutions. The crystals are re-dissolved in acidified isopropanol and the resulting purple solution is measured spectrophotometrically. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test material.
After 16-hour incubation of corneal epithelium, the units are incubated for 3 h in 300 μl/well in a 24 well plate of 1mg/ml MTT solution at 37 °C. The solution is then removed and replaced with 1500 μl/well of isopropanol, with further 2 h incubation at room temperature under medium speed shaking.
The absorbance at 570 nm is measured with a microplate reader (Tecan modello Sunrise remote). The absorbance values are corrected by subtracting the reading of the blanks, with the diluent only.
The results are expressed in terms of viability:
% of cell viability = [OD(570 nm ) test product / mean OD(570 nm) negative control] × 100
Acceptance criteria of method
for negative control (CN): the mean OD value of the 3 tissue has to be ≥ 0.7, the standard deviation has to be ≤ 18 %
for positive control (CP): the viability mean (expressed as % of the NC) has to be ≤ 50 %, the standard deviation has to be ≤ 18 %
for the sample : the standard deviation has to be ≤ 18 %
Expression of results
Mean cell viability is predictive of irritant ocular potential of the sample.
Criteria for in vitro interpretation Classification
Mean tissue viability ≤ 50 % classified
Mean tissue viability > 50 % not classified
Results and discussion
In vitro
Results
- Irritation parameter:
- other: mean cell viability in %
- Run / experiment:
- average of 3 replicates
- Value:
- 100.7
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- Sample % mean cell viability (ds) after 1 h treatment and 16 h incubation Comment
test substance 100.7 (± 1.2) Not irritant
SLS 0.5 % 1.2 (± 0.7) Irritant
negative control (CN) 100.0 (± 2.8) Not irritant
Applicant's summary and conclusion
- Interpretation of results:
- other: not irritant according to the CLP Regulation (EC 1272/2008)
- Conclusions:
- Not irritant to the eye under test conditions.
- Executive summary:
Method
In vitro assay using a reconstituted 3D human corneal epithelium. Test sample was applied on 3 replicates for 1 hour at room T. Then, the substance was removed and tissues were incubated for 16 h at 37 °C and 5 % CO2. After incubation, evaluation of survival was done using the MTT assay and measuring absorbance at 570 nm. Based on absorbance values, a % of cell viability was derived.
Phosphate buffer was used as negative control; 0.5 % solution in water of SLS was used as positive control. Controls were run in 3 replicates.
Results
Negative and positive controls were both valid. The mean cell viability of test substance was 100.7 ± 1.2 %, thus above the threshold of 50 %. Accordingly, the substance was considered as not irritant to the eye.
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