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EC number: - | 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
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
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 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:
- 18 June 2019
- Deviations:
- no
- GLP compliance:
- yes
Test material
- Reference substance name:
- Constituent C25H31N | Nonyl (branched) substituted - N-Phenyl-1-naphthylamine
- Molecular formula:
- C25H31N
- IUPAC Name:
- Constituent C25H31N | Nonyl (branched) substituted - N-Phenyl-1-naphthylamine
- Reference substance name:
- Constituents C34H49N | Di-nonyl (branched)- substituted N-Phenyl-1-naphthylamine
- Molecular formula:
- C34H49N
- IUPAC Name:
- Constituents C34H49N | Di-nonyl (branched)- substituted N-Phenyl-1-naphthylamine
- Test material form:
- liquid
- Details on test material:
- Name of test item: XPDL 958
Test item No.: 20/0205-1
Batch identification: 0021851006
CAS No.: 63451-49-0
Purity: 100% UVCB *; additionally, an analytical characterization was conducted (No. 20L00034).
Homogeneity: The test item was homogeneous by visual inspection.
Storage stability: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Expiry date: November 18, 2021
Storage conditions: Room temperature
Physical state / color: Liquid, viscous/ brown to red
Constituent 1
Constituent 2
Test animals / tissue source
- Species:
- human
- Details on test animals or tissues and environmental conditions:
- TEST SYSTEM
Three-dimensional human cornea model
The EpiOcularTM model (OCL-200) is a three-dimensional, non-keratinized tissue construct
composed of normal human-derived epidermal keratinocytes used to model the human corneal
epithelium (compare Figure 1). The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell
culture inserts (MILLICELLs, 10 mm ∅) and are available commercially as kits
(EpiOcular™ 200) containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Tissue Lot Number: 30669 (Certificate of Analysis see Appendix)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Test system
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes
- Amount / concentration applied:
- see details on study design
- Duration of treatment / exposure:
- see details on study design
- Observation period (in vivo):
- see details on study designsee details on study design
- Duration of post- treatment incubation (in vitro):
- see details on study design
- Number of animals or in vitro replicates:
- see details on study design
- Details on study design:
- ANALYSES
No analysis of test-substance preparation was performed because the test substance was applied undiluted.
EXPERIMENTAL PROCEDURE
Pretest for direct MTT reduction
The direct reduction of MTT by a test substance interferes with the color density produced by
metabolic capacity of the tissue and would falsify the test results.
In order to assess the ability of the test material to reduce MTT directly, a pretest (experimental
conduct in accordance with GLP, but without a GLP status) was performed as described below.
The test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark
at about 37°C for 3 hours. A negative control (deionized water) was tested concurrently.
If the color of the MTT solution or (in case of water-insoluble test substances) the border to the
water phase turned blue / purple, the test substance was presumed to reduce MTT directly.
In case of direct MTT reduction, two freeze-killed control tissues (KC) were treated additionally
with each the test article and the negative control in the same way as described in section 3.6.
Based on the result of the pretest, it was judged that application of killed control tissues is
necessary.
Basic procedure
Several test substances were tested in parallel within the present test (test no. 122) using the
same control tissues (NC and PC).
Two tissues were treated with each the test substance, the PC and the NC.
In addition, two killed tissues were used for each the test substance and the NC to detect direct
MTT reduction.
There are two separate protocols for liquids and solids differing in exposure time and postincubation
period. Due to the physical condition of the test substance, the protocol for liquids
was applied.
Pre-incubation of the tissues
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with
1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour, the
pre-incubation medium was replaced with fresh medium, and preconditioning continued in the
incubator at standard culture conditions for 16 – 24 hours.
Pretreatment of the tissues
After pre-incubation, the tissues were pretreated with 20 μL PBS in order to wet the tissue
surface. The tissues were incubated at standard culture conditions for 30 minutes.
Application of the test substance
Using a pipette, fifty microliters (50 μL) undiluted liquid test substance were applied covering
the whole tissue surface.
Control tissues were applied concurrently with 50 μL sterile deionized water (NC, NC KC) or
with 50 μL methyl acetate (PC) or test substance (KC).
After application, the tissues were placed into the incubator until the total exposure time of
30 minutes was completed.
Removal of the test substance and postincubation period
In order to remove the test substance, the tissues were washed with sterile PBS. For this
purpose, the tissues were immersed and swiveled three times in each of three beakers filled
with PBS. The washing procedure was intensified by careful wiping with UltraClean™ LASIK
Expanded Eye Spears (Beaver Visitec International) to remove as much compound residues
as possible. Washed tissues were immersed immediately into 12-well plates pre-filled with
5 mL/well pre-warmed medium (post-soak immersion) to remove further residual test
substance. After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper
and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium.
Subsequently, the tissues were incubated at standard culture conditions for 2 hours (postincubation
period).
MTT incubation
After the post-incubation period, the assay medium was replaced with 0.3 mL MTT solution,
and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT incubation.
The formazan that was metabolically produced by the tissues was extracted by incubation of
the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts
was spectrophotometrically determined. Blank values were established of 4 microtiter wells
filled with isopropanol for each microtiter plate.
Data evaluation
Table(s) of measured parameters that are presented in the report were produced using a
computer-based tabular calculation software. The mean and individual data were not always
rounded, but the significant digits were produced by changing the display format. As a
consequence, calculation of mean values by using the individual data presented in the report
will in some instances yield minor variations in value.
Principle
The OD570 values determined for the various tissues are a
measure of their viability. The ratio of the OD570 of tissues
treated with the test material and the mean OD570 values of the
NC (percent of control) is used for evaluating whether a test
material was an irritant.
Calculation of individual and mean optical densities
The corrected measured OD570 value for each individual tissue
was calculated by subtracting the mean blank value of the
respective microtiter plate from the respective individual tissue
OD570 value. The mean OD570 for a test group of two tissues
treated in the same way was calculated.
Application of measurements using killed control tissues
In case of direct MTT reduction by the test substance, the OD570
values measured in the freeze-killed control tissues (KC) will be
used to correct the mean OD570 of the tissues treated with the
test substance (mean corrected OD570 KC). Since a killed tissue
might still have a residual enzyme activity that is able to produce
some formazan net, OD570 KC is calculated by subtracting the
OD570 KC of the NC from the OD570 KC of the test substance.
In case the net OD570 KC is greater than zero, it is subtracted
from the respective mean OD570 to result in the mean corrected
OD570 KC. The mean corrected OD570 KC represents the
formazan production linked to the tissue viability and therefore
indicates the cytotoxic potency of the test substance.
Tissue viability
The quantification of tissue viability is presented as ratio of the
mean OD570 (or mean corrected OD570 KC if applicable) divided
by the respective OD570 NC value in percent.
ACCEPTANCE CRITERIA
In case one of the acceptance criteria below was not met, repetition of the test was considered.
Barrier function and Quality control (QC)
The supplier demonstrates that each batch of the model used
meets the defined production release criteria. MatTek
determines the ET50 (min) value following exposure to 100 μL
of 0.3% Triton X-100 for each EpiOcular™ EIT (OCL-200)
batch. The ET50 must fall within an established range based on
a historical database of results.
The following acceptability range (upper and lower limit) for the
ET50 is established by the supplier as described in the cited
OECD Guideline.
Lower acceptance limit: ET50 = 12.2 min
Upper acceptance limit: ET50 = 37.5 min
EpiOcular QC (OCL-200) batch release see Appendix
Acceptance criteria for the NC
The absolute OD570 of the NC tissues in the MTT test is an
indicator of tissue viability obtained in the testing laboratory
after the shipping and storing procedure and under specific
conditions of the assay. Tissue viability is acceptable if the
mean OD570 of the NC is > 0.8. The mean OD570 of the NC
should not exceed 2.8.
Acceptance criteria for the PC
Methyl acetate used as PC usually leads to a tissue viability of
approx. 25%. A viability of < 50% is acceptable.
Acceptance criteria for tissue variability
Two tissues were treated under the same conditions.
A variability between the tissues is considered to be acceptable
if the relative difference of the viability is < 20%.
Acceptance criteria for the KC
The OD570 of the killed control tissues treated as negative
control should be ≤ 0.35. The value for direct MTT reduction of
a test substance should be ≤ 30% of the NC.
EVALUATION OF RESULTS
The eye irritation potential of the test material is predicted from the mean relative tissue
viabilities compared to the negative control tissues treated concurrently with sterile water. A
chemical is considered as "non-irritant” (no UN GHS Category) if the mean relative tissue
viability with a test material is greater than 60%.
A single test composed of at least two tissue replicates should be sufficient for a test chemical
when the result is unequivocal. However, in case of borderline results such as non-concordant
replicate measurements and/or mean percent tissue viability equal to ± 5% of the cut-off value,
a second test should be considered as well as a third one in case of discordant results between
the first two tests.
The following decision criteria apply:
Mean tissue viability (% of negative control)
<= 60 % No prediction can be made
> 60 % No UN GHS Category
A “borderline“ evaluation (60 ± 5%) was determined statistically using historic BASF data and
hence considers the variance of the test method. This evaluation is confirming the borderline
range provided in OECD Guideline 492.
HISTORIC CONTROL DATA
Historic control values of negative and positive controls collected over an appropriate period
are presented in section 4.2. These data demonstrate the reproducibility of results and
robustness of the procedures. They are used to derive suitable acceptance criteria (see
section 3.7.) for the test system.
Results and discussion
In vitro
Resultsopen allclose all
- Irritation parameter:
- mean percent tissue viability
- Remarks:
- test substance, viable tissues
- Run / experiment:
- mean
- Value:
- 96.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- mean percent tissue viability
- Remarks:
- test substance after KC correction
- Run / experiment:
- mean
- Value:
- 94.9
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- mean percent tissue viability
- Remarks:
- negative control, viable tissue
- Run / experiment:
- mean
- Value:
- 100
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- mean percent tissue viability
- Remarks:
- positive control, viable tissue
- Run / experiment:
- mean
- Value:
- 18.9
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Other effects / acceptance of results:
- Slight compound residues (brown-red colored) remained on the KC tissues treated with the
test substance after the washing procedure.
Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in
parallel. The results of the KC tissues indicate an increased MTT reduction (relative mean
viability 1.6 % of NC). Thus, for the test substance the final relative mean viability is given after
KC correction.
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Based on the results observed and by applying the evaluation criteria it was concluded that XPDL 958 does not show an eye irritation potential in the EpiOcular™ in vitro eye irritation test under the test conditions chosen.
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