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EC number: 209-013-8 | CAS number: 552-45-4
- 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
Eye irritation
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-reference
- Reason / purpose for cross-reference:
- other: part of a testing Strategy
Reference
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- other: part of a testing strategy
- 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:
- 26 July 2013
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Species:
- cattle
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: freshly slaughtered cattle; supplier: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals: age of the animals: minimum 12 months, maximum 60 months
- indication of any existing defects or lesions in ocular tissue samples: Corneas free of defects (opacity, scratches, pigmentation etc.) - Vehicle:
- unchanged (no vehicle)
- Remarks:
- de-ionized
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL of de-ionized water
POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 μL 100% ethanol / 100% dimethylformamide (positive controls, PC1 / PC2) - Duration of treatment / exposure:
- 4 hours
- Number of animals or in vitro replicates:
- 3
- Details on study design:
- SELECTION AND PREPARATION OF CORNEAS: Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32°C for at least 1 hour. After the equilibration period, the medium in both chambers was replaced by fresh pre-warmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer.
QUALITY CHECK OF THE ISOLATED CORNEAS: Any corneas that showed macroscopic tissue damage or an opacity value < 550 opacity units were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups.(According to OECD TG 437, corneas that have an opacity value >7 or equivalent for the opacitometer and cornea holders used after an initial one-hour equilibration period are to be discarded. In the test facility´s opacitometer in combination with the used cornea holder set 2013-22 and 2013-24, the maximal initial opacity of >7 arises from I= 550 lux with Io= 641 lux).
NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: Yes
POSITIVE CONTROL USED: Yes
APPLICATION DOSE AND EXPOSURE TIME: Application of 750 µL of the 20% (w/v) test-substance preparation, 4 hours exposure time
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3times.
- POST-EXPOSURE INCUBATION: no
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Yes.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader: Yes, OD490, SunriseTM Absorbance Reader.
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA: amendment to TG (refinement of decision criteria).
IVIS: < 1.5, Prediction: No classification for eye irritation.
IVIS: 1.5 – 4.5, Prediction: Borderline.
IVIS: > 4.5, < 45, Prediction: No prediction can be made for eye irritation, further testing with another suitable method is required.
IVIS: 45 - 65, Prediction: Borderline.
IVIS: > 65, Prediction: Ocular corrosive or severe irritant.
The “borderline“ evaluation (IVIS 3.0 ± 1.5 and 55.0 ± 10.0) was statistically determined by using historic test facility data and takes the test facility specific variance of the test method into account. This evaluation is an amendment to the evaluation provided in OECD Guideline 437 - Irritation parameter:
- in vitro irritation score
- Run / experiment:
- Run 1, mean of three single corneas
- Value:
- 5
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Interpretation of results:
- other: not identified as corrosive or severe irritant.
- Conclusions:
- Based on the results of the BCOP assay, the test substance was not identified as corrosive or severe irritant.
- Executive summary:
The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL undiluted test substance to the epithelial surface of isolated bovine corneas.
Three corneas were treated with the test substance for 10 minutes followed by a 2-hour post-incubation period.
In addition to the test substance, a negative control (NC; deionized water) and two positive controls (PC1 / PC2; 100% ethanol / 100% dimethylformamide) were applied to three corneas each.
Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance.
The mean IVIS obtained in the BCOP test was 5.1, therefore the substance was not identified as corrosive or severe irritant.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
- Version / remarks:
- 28 July 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- α-chloro-o-xylene
- EC Number:
- 209-013-8
- EC Name:
- α-chloro-o-xylene
- Cas Number:
- 552-45-4
- Molecular formula:
- C8H9Cl
- IUPAC Name:
- 1-(chloromethyl)-2-methylbenzene
- Test material form:
- liquid
Constituent 1
Test animals / tissue source
- Species:
- human
- Strain:
- other: normal human derived epidermal keratinocytes
- Details on test animals or tissues and environmental conditions:
- Description of the cell system used: Reconstructed human cornea-like epithelium
- Model used: EpiOcular™ model (OCL-200)
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Certificate of authenticity: provided by the supplier
- Verification of tissue functionality and quality (performed by the supplier):
Tissue viability: acceptance criteria met
Barrier function: acceptance criteria met
Sterility: no contamination
Test system
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): ca. 50 μL of the undiluted test substance
NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL
POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL, undiluted - Duration of treatment / exposure:
- 6 hours
- Duration of post- treatment incubation (in vitro):
- 18 hours
- Number of animals or in vitro replicates:
- 2
- Details on study design:
- TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation: 37°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were immersed and swiveled three times in each of three beakers filled with sterile PBS.
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
A direct interference was not observed.
NUMBER OF REPLICATE TISSUES PER TEST CHEMICAL AND CONTROLS: 2
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm, without reference filter
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to the eye if the mean relative tissue viability with a test material is less than 55%.
- The test substance is considered to be non- irritant to the eye if the mean relative tissue viability with a test material is greater than 65%.
- In case of borderline results such as non-concordant replicate measurements and/or mean percent tissue viability of 55 - 65%, a second test should be considered as well as a third one in case of discordant results between the first two tests.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 492: 4The „borderline“-evaluation (60 ± 5%) was determined statistically using historic data of the test facility and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 492.
ACCEPTANCE CRITERIA
- Negative control: Tissue viability is acceptable if the mean OD570 of the negative control (NC) is ≥ 0.8. The mean OD570 of the NC should not exceed 2.5.
- Positive control: Methyl acetate used as positive control (PC) usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
- Variability: Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the difference of the viability is ≤ 20%.
HISTORICAL DATA (see table 1)
Results and discussion
In vitro
Results
- Irritation parameter:
- other: mean viability [%]
- Run / experiment:
- Run 1, mean of 2 replicate tissues
- Value:
- 51.3
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
Applicant's summary and conclusion
- Interpretation of results:
- other: irritant
- Conclusions:
- Using the currently available methods a single in vitro assay is not sufficient to cover the full range of eye irritating potential. In combination with the BCOP assay, which identified the test substance as not corrosive or severe irritant, the test substance is classified as GHS Cat. 2.
- Executive summary:
The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50 μL undiluted test substance to a reconstructed three-dimensional, human
cornea model (EpiOcular™).
Two EpiOcular™ tissues were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period.
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.
The following results were obtained in the EpiOcular™ eye irritation assay: The test substance is not able to directly reduce MTT.
The relative mean viability of the tissues treated with the test substance was 51.3%.
Based on the results of the BCOP and EpiOcular Tests and by applying the evaluation criteria described in section 3.10, it was concluded that 2-Methylbenzyl chloride shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.
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