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EC number: 951-779-7 | CAS number: -
- 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:
- Between 14 February and 29 April 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study performed according to OECD Guideline 431 without any deviation.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 020
- Report date:
- 2020
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
- Version / remarks:
- 18 June 2019
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 14 October 2019
Test material
- Reference substance name:
- Absolute of Nicotiana tabacum (Solanaceae) obtained from leaves by organic solvent treatment and subsequent ethanol extraction
- EC Number:
- 951-779-7
- Molecular formula:
- Not relevant, UVCB substance
- IUPAC Name:
- Absolute of Nicotiana tabacum (Solanaceae) obtained from leaves by organic solvent treatment and subsequent ethanol extraction
- Test material form:
- liquid
- Details on test material:
- - Physical state: Liquid
- Storage condition of test material: room temperature, darkness (Optimal temperature at 20°C)
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: foreskin
- Justification for test system used:
- Following the REACH bottom-up strategy described in the ECHA R.7a guidance, after the positive result obtained in the SkinEthic RHE® model method for skin irritation, the EpiCS™ RHE Model method was used to assess skin corrosion.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 0.6 cm² reconstituted epidermis and four additional killed control tissues (EpiCS™, supplied by CellSystems)
- Tissue batch number(s): 100-AJ0806-1 (living tissues) / 100-AI1214-1 (killed control tissues)
- Expiration date: no data
- Date of received: 28 April 2020 for the living tissues and defrozen on 29 April 2020 for the killed control tissues
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes (room temperature) and 1 hour (37°C +/- 1°C)
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 mL DPBS, 20 times
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL concentration for the skin sample and MTT assay medium for the 4 tissues used for the non-specific control
- Incubation time: 3 hours at 37 +/- 1°C and 5% CO2.
- Spectrophotometer: ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
- Wavelength: 570 nm
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: viability % = OD test item/OD negative control x 100. For each tissue, OD values and calculated percentage cell viability data for the test item, positive and negative controls, were calculated.
- As the test item was identified as producing direct MTT reduction, the true tissue viability is then calculated as follows: True viability % = [(OD of living tissues exposed to test item - OD of killed tissues exposed to test item) / OD of living tissues exposed to negative control] x 100
NUMBER OF REPLICATE TISSUES: 2
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of solution of MTT at 1 mg/mL. A purple suspension was observed after 1 hour of incubation between 37.1°C and 37.4°C, 5% CO2.
> Therefore, the test item was identified as a direct MTT reducer. Four additional killed control tissues were added to the study. These four control tissues (two for 3-minute treatment and two for 1-hour treatment) were incubated with culture medium instead of MTT solution during the MTT incubation step to generate a non-specific colour control (NSC control).
SPECTRAL ANALYSIS OF THE TEST ITEM IN ISOPROPANOL
The spectral properties at 570 nm of test item in isopropanol were checked by adding 50 µL of the test item to 2 mL of isopropanol. A yellow solution was obtained after 3 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD was 0.067 which is lower than 0.08.
> Therefore, the test item will not interfere with the MTT assay and there is no need to add non-specific coloration controls to the study.
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2
PREDICTION MODEL / DECISION CRITERIA
- Step 1: The test substance is considered to be classified to skin as follows if the viability is:
< 50% after 3 min exposure = Corrosive
≥ 50% after 3 min exposure AND < 15% after 60 min exposure = Corrosive
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure = Non-corrosive
- Step 2: The test substance is subcategorised for items identified as corrosive in step 1:
1A if viability is < 15% after 3 min exposure
1B/1C if viability is ≥ 15% after 3 min exposure - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): undiluted
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): undiluted
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): undiluted - Duration of treatment / exposure:
- 3 minutes and 1 hour
- Duration of post-treatment incubation (if applicable):
- not applicable
- Number of replicates:
- 2
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Remarks:
- corrected with 4 killed human skin model surfaces to generate non-specific MTT reduction
- Run / experiment:
- 3 minutes
- Value:
- 84.47
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks:
- 58.06%
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- % tissue viability
- Remarks:
- corrected with 4 killed human skin model surfaces to generate non-specific MTT reduction
- Run / experiment:
- 1 hour
- Value:
- 86.98
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks:
- 0.5 %
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the epidermis skins treated with the test item were 84.47% and 86.98%, versus 58.06% and 0.5%, respectively, with the positive control item (potassium hydroxide 8N).
- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: Yes, four additional killed control tissues were used to generate non-specific MTT reduction
- Colour interference with MTT: none.
DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 431.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the negative control OD of the 2 replicates is >= 0.8 and <= 2.8 for every exposure time. As the extract was diluted at 1:3 just before the OD mesure, the acceptability criteria should be in the range >= 0.3 and <=0.9 for the negative control (values 0.667 for 3-minute exposure and 0.599 for 1-hour exposure).
- Acceptance criteria met for positive control: yes, the mean tissue viability of the 2 replicates exposed for 1 hour are < 20% (0.50 %)
- Acceptance criteria met for variability between replicate measurements: yes, in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates was < 30%.
- Acceptance criteria of range of historical values: The mean viability of epidermises treated with positive control during 3 minutes was 58.06%, which is in the range of historical data (0.40% and 85.12%) for 3-minute exposure and the mean viability of epidermises treated with positive control during 1-hour was 0.50%, which is in the range of historical data (0.00% and 1.32%) for 1-hour exposure.
Any other information on results incl. tables
Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls
Tissue | Exposure Period | OD | Mean OD/ disc (#) | Mean OD / Product | Viability (%) | Mean viability (%) | Viability difference between replicates (%) |
Negative Control | 3 Minutes | 0.685 0.697 0.713 | 0.698 | 0.667 | 104.73 | 100.00 | 4 9.5 |
0.644 0.635 0.627 | 0.635 | 95.27 | |||||
60 Minutes | 0.595 0.656 0.643 | 0.631 | 0.599 | 105.34 | 100.00 | 10.7 | |
0.554 0.557 0.590 | 0.567 | 94.66 | |||||
Positive Control | 3 Minutes | 0.319 0.347 0.345 | 0.337 | 0.387 | 50.56 | 58.06 | 15.0 |
0.450 0.435 0.428 | 0.437 | 65.57 | |||||
60 Minutes | 0.003 0.002 0.004 | 0.003 | 0.003 | 0.50 | 0.50 | 0.0 | |
0.002 0.003 0.003 | 0.003 | 0.50 | |||||
Test Item | 3 Minutes | 0.625 0.624 0.620 | 0.623 | 0.576 | 93.47 | 86.42 | 14.1 |
0.521 0.542 0.526 | 0.529 | 79.37 | |||||
60 Minutes | 0.578 0.611 0.602 | 0.597 | 0.532 | 99.67 | 88.81 | 21.7 | |
0.459 0.477 0.465 | 0.467 | 77.96 | |||||
Test item killed tissues | 3 minutes | 0.017 0.017 0.016 | 0.016 | 0.013 | 2.40 | 1.95 | 0.9 |
0.010 0.011 0.011 | 0.010 | 1.50 | |||||
60 minutes | 0.012 0.013 0.010 | 0.012 | 0.011 | 2.00 | 1.84 | 0.3 | |
0.009 0.009 0.011 | 0.010 | 1.67 | |||||
Test item corrected | 3 minutes | 84.47 |
| ||||
60 minutes |
| 86.98 |
|
OD: optical density
#: mean of 3 values
Applicant's summary and conclusion
- Interpretation of results:
- Category 2 (irritant) based on GHS criteria
- Conclusions:
- Under the experimental conditions of this study, the test substance is not classified for skin corrosion according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS. Based on the result of the in vitro skin irritation study, the test substance is therefore classified as Skin irritant Category 2 (H315: Causes skin irritation) according to CLP and GHS
- Executive summary:
An in vitro skin corrosion study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiCS reconstructed human epidermis model.
The test item TOBACCO ABSOLUTE was applied as supplied, at the dose of 50 µL, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour, followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Additionally, 4 killed Human skin model surfaces were treated under the same experimental conditions in order to generate non-specific MTT reduction.
3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 84.47% and 86.98%, versus 58.06% and 0.5%, respectively, with the positive control item (potassium hydroxide 8N).
All the acceptability criteria were met and the quality criteria required for acceptance of results in the test were satisfied:
- the negative control OD of the 2 replicates is >= 0.8 and <= 2.8 for every exposure time. As the extract was diluted at 1:3 just before the OD mesure, the acceptability criteria should be in the range >= 0.3 and <=0.9 for the negative control (values 0.667 for 3-minute exposure and 0.599 for 1-hour exposure).
- the mean tissue viability of the 2 replicates exposed for 1 hour are < 20% (0.50 %)
- Variability between replicate measurements in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates was < 30%.
- The mean viability of epidermises treated with positive control during 3 minutes was 58.06%, which is in the range of historical data (0.40% and 85.12%) for 3-minute exposure and the mean viability of epidermises treated with positive control during 1-hour was 0.50%, which is in the range of historical data (0.00% and 1.32%) for 1-hour exposure.Under the experimental conditions of this study, the test substance is not classified for skin corrosion according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS. Based on the result of the in vitro skin irritation study, the test substance is therefore classified as Skin irritant Category 2 (H315: Causes skin irritation) according to CLP and GHS.
This study is considered as acceptable and satisfies the requirement for skin corrosion endpoint.
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