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EC number: 264-776-4 | CAS number: 64325-78-6
- 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:
- weight of evidence
- Study period:
- 2017-11-22 to 2018-02-12
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
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:
- Adopted 28 July 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- N-benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxyadenosine
- EC Number:
- 264-776-4
- EC Name:
- N-benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxyadenosine
- Cas Number:
- 64325-78-6
- Molecular formula:
- C38H35N5O6
- IUPAC Name:
- N-benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxyadenosine
- Test material form:
- solid
Constituent 1
Test animals / tissue source
- Species:
- human
- Details on test animals or tissues and environmental conditions:
- - Justification of the test method: This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years.
- Description of the cell system used: The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium that is morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.
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): 50 mg - Duration of treatment / exposure:
- 6 ± 0.25 h
- Observation period (in vivo):
- n.a.
- Duration of post- treatment incubation (in vitro):
- Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 1 °C - Number of animals or in vitro replicates:
- 2 tissues per dose group
- Details on study design:
- - Details of the test procedure used:
Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. The EpiOcular™ tissues were then transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h at 37 +/- 1 °C, 5.0% CO2 / 95% air. Then the tissues were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air for 16 - 24 h.
After the overnight incubation the tissues were pre-treated with 20 µL of DPBS and incubated for 30 ± 2 min in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye. Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. The test item was applied on the tissues placed on a sterile surface. Start time was recorded with dosing of the first tissue and occurred sequentially for the other tissues, e.g. in one-minute intervals. After dosing, the tissues were placed back into the culture medium. Then the 6-well plate(s) were incubated for 6 ± 0.25 h at 37 +/- 1 °C, 5.0% CO2 / 95% air until the 6 ± 0.25 h of the first dosed tissue was over. At the end of the exposure period, the test item and control substances were removed by extensively rinsing the tissue with DPBS, occurring also in sequential order, e.g. in one-minute intervals. Excess DPBS was removed by decanting the insert and blotting the bottom with a blotting paper. After rinsing, the tissues were transferred to and immersed in a pre-prepared 12-well “post-soak plate“, containing 2 mL fresh pre-warmed assay medium per well and incubated for 25 ± 2 min at room temperature. Afterwards, the tissues were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on a blotting paper. The tissues were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 18 ± 0.25 h at 37 +/- 1 °C, 5.0% CO2 / 95% air.
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a pre-prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h +/- 10 min at 37 +/- 1 °C, 5.0% CO2 / 95% air.
After the 3 h MTT incubation period the inserts were removed, with the bottom of the inserts blotted on blotting paper, and then transferred into new 6-well “extraction plates“, containing 2 mL of isopropanol to extract the formazan only from the bottom of the tissues to avoid possible contamination of test material. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out after storage overnight in the dark at 2 - 8 °C. At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item.
Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings. For each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.
- RhCE tissue construct used, including batch number:
EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek), consisting of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.
The EpiOcular™ tissues were provided as kits (e.g. OCL-200-EIT; MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing 24 inserts with EpiOcular™ tissues on agarose (Lot No.: 27017),
1x bottle EpiOcularTM assay medium (Lot No.: 121117ISA),
1x bottle Ca2+/Mg2+-free DPBS buffer (Lot No.: 092817MGKA)
- Doses of test chemical and control substances used:
1. Negative Control: 50 µL Aqua dest. (Sigma, Lot No. RNBF7110)
2. Positive Control: 50 µL methyl acetate (CAS No. 79-20-9 (Lot No.: S6943111)
3. Test Item: 50 mg
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
Exposure: 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2/95% air.
Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 1 °C, 5.0% CO2/95% air
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
See section "Pre-experiments" in box "Any other information on materials and methods incl. tables"
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per group
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm ± 30 nm
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
Mean tissue viability (% negative control) <= 60 %: Irritant (I): UN GHS “Category 1” or “Category 2”
Mean tissue viability (% negative control) > 60%: Non-Irritant (NI): UN GHS “No Category”
Results and discussion
In vitro
Results
- Irritation parameter:
- other: Relative Tissue Viability (%)
- Run / experiment:
- Mean of replicates
- Value:
- 44.9
- Vehicle controls validity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Other effects / acceptance of results:
- The test item showed irritant effects. The mean relative tissue viability (% negative control) was < 60% (44.9%). For detailed information please refer to Table 1 in box "Any other information on results incl. tables".
Applicant's summary and conclusion
- Interpretation of results:
- other: UN GHS Category 1 or Category 2
- Conclusions:
- In this study under the given conditions the test item showed irritant effects. The test item is classified as “irritant“ in accordance with UN GHS “Category 1” or “Category 2”.
- Executive summary:
In the present study the eye irritation potential of N-Benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxyadenosine (100% purity) was analysed according to OECD 492 using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. Hereby, 50 mg of the test item was applied directly atop the EpiOcular™ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 6-hour exposure and 18-hour post-incubation period and compared to those of the concurrent negative controls. The test item showed no reduction of MTT as compared to the control. Therefore, NSMTT was determined to be 0%.
The test item showed irritant effects. The mean relative tissue viability of two replicates (% negative control) was < 60% (44.9 %). Therefore, the test item is considered to be irritating to the eye in accordance with UN GHS “Category 1” or “Category 2”.
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