Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 203-192-6 | CAS number: 104-29-0
- 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
![](https://echa.europa.eu./o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From March 4th to 10th, 2014
- 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:
- 2 014
- Report date:
- 2014
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- July 22nd, 2010
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Chlorphenesin
- EC Number:
- 203-192-6
- EC Name:
- Chlorphenesin
- Cas Number:
- 104-29-0
- Molecular formula:
- C9H11ClO3
- IUPAC Name:
- 3-(4-chlorophenoxy)propane-1,2-diol
- Test material form:
- solid: particulate/powder
Constituent 1
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark.
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Details on animal used as source of test system:
- SOURCE ANIMAL
- Source: adult human - Justification for test system used:
- Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.
Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man. - Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Tissues (0.38 cm2) supplied by SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 14-EKIN-007
- Delivery date: 04 March 2014
- Date of initiation of testing: 04 March 2014
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C
PRE-TEST PROCEDURE
Assessment of Direct Test Item Reduction of MTT
-MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.
- Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test item is checked for the ability to directly reduce MTT according to the following procedure: 10 mg of the test item was added to 2 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.
Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: yes
Temperature Indicator Color Satisfactory: yes
Agar Medium Color Satisfactory: yes
2 ml of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.
MAIN TEST
- Application of Test Item and Rinsing (Day 1)
2 ml of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5µl of sterile distilled water was topically applied to the epidermal surface in order to improve further contact between the test item and the epidermis. 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 µl of DPBS served as the negative controls and triplicate tissues treated with 10 µl of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7-Minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca 2+ and Mg2+. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
- MTT Loading/Formazan Extraction (Day 3)
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 ml of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 °C for possible inflammatory mediator determination.
2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 ml micro tubes containing 500 µl of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
- Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200µl samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200µl of acidified isopropanol alone was added to the two wells designated as 'blanks'. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: at the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca2+ and Mg 2+. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 ml of a 0.3 mg/mL MTT solution, freshly prepared in assay medium
- Incubation time: 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 ml micro tubes containing 500 μl of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
- Wavelength: 562 nm
- Filter: without reference filter
- Filter bandwidth:none
NUMBER OF REPLICATE TISSUES: three replicates
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
No possible interference of the test item to directly reduce MTT was checked according to the procedure reported in pre-test procedure.
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after 15-Minute exposure period followed by the 42-Hour post-exposure incubation exposure is lower than or equal to 50 %.
- The test substance is considered to be non-irritant to skin if the tissue viability after 15-Minute exposure period followed by the 42-Hour post-exposure incubation exposure is greater than 50 %.
ACCEPTANCE CRITERIA
- Acceptance criteria met for negative control: Phosphate Buffered Saline Dulbecco's (PBS): Mean OD562 for the negative control treated tissues was ≥ 0.6, and the standard deviation value of the percentage viability is ≤ 18%.
- Acceptance criteria met for positive control: Sodium Dodecyl Sulphate (SDS) 5% w/v aqueous solution: Mean tissue viability for the positive control treated tissues was ≤ 40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤ 18%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤ 18%. - Control samples:
- other: negative and positive controls (3 replicates for each)
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface previous treated with 5 μL of sterile distilled water in order to improve further contact between the test item and the epidermis.
VEHICLE: no vehicle
NEGATIVE CONTROL
- Amount(s) applied (volume or weight):10 µL of Phosphate Buffered Saline Dulbecco's (PBS)
POSITIVE CONTROL
- Amount(s) applied (volume or weight):10 μL of Sodium Dodecyl Sulphate (SDS) 5% w/v aqueous solution - Duration of treatment / exposure:
- 15 minutes.
- Duration of post-treatment incubation (if applicable):
- 42 hours at 37 °C and at 5% CO2 in air.
- Number of replicates:
- 3 replicates for negative control, positive control and test item
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean of three replicates
- Value:
- ca. 82.7
- Vehicle controls validity:
- not applicable
- Remarks:
- no vehicle used
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- - RESULTS:
The relative mean viability of the test item treated tissues was 82.7% after 15 minutes exposure period and 42 hours of post-exposure incubation period.
- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Phosphate Buffered Saline Dulbecco's (PBS): The mean OD562 for the negative control treated tissues was 0.915 ( ≥ 0.6) and the standard deviation value of the percentage viability was 3.3 % (≤ 18 %). The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: Sodium Dodecyl Sulphate (SDS) 5% w/v aqueous solution: the relative mean tissue viability for the positive control treated tissues was 7.7% (≤ 40 %) relative to the negative control treated tissues and the standard deviation value of the percentage viability was 2.3% (≤ 18%). The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: the standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 4.0 % (≤ 18 %). The test item acceptance criterion was therefore satisfied.
Any other information on results incl. tables
For the test item the relative mean tissue viabilities obtained after the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD562of test item/ mean OD562of negative control)*100
Item |
OD562of tissues |
Mean OD562of triplicate tissues |
±SD of OD562 |
Relative individual Tissue viability(%) |
Relative mean viability (%) |
±SD of Relative mean viability (%) |
Negative Control |
0.914 |
0.915 |
0.030 |
99.9 |
100 |
3.3 |
0.886 |
96.8 |
|||||
0.945 |
103.3 |
|||||
Positive Control |
0.095 |
0.071 |
0.021 |
10.4 |
7.7 |
2.3 |
0.059 |
6.4 |
|||||
0.059 |
6.4 |
|||||
Test item |
0.753 |
0.757 |
0.036 |
82.3 |
82.7 |
4.0 |
0.795 |
86.9 |
|||||
0.723 |
79.0 |
Applicant's summary and conclusion
- Interpretation of results:
- other: not classified as skin irritant, according to the CLP Regulation (EC n.1272/2008)
- Conclusions:
- The test item was classified as non-irritant. The following classification criteria apply:
- EU DSD & CLP: Not classified for Irritation.
- UN GHS: Not classified for Irritation (category 3 can not be determined) - Executive summary:
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point can be used to either confirm a non-irritant result or will be used to override the non-irritant result.
Method:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 562 nm.
Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).
Results:
The relative mean viability of the test item treated tissues was 82.7% after the 15-Minute exposure period and 42 hours post-exposure incubation period. The quality criteria required for acceptance of the results in the test were satisfied.
Conclusion
The test item was classified as non-irritant. The following classification criteria apply:
- EU DSD & CLP Not classified for Irritation.
- UN GHS Not classified for Irritation (category 3 can not be determined).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
![ECHA](/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/echa_logo.png)