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EC number: 950-170-3 | 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 sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Experimental start date: 17 November 2017 Experimental completion date: 21 December 2017
- 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 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
- Deviations:
- yes
- Remarks:
- Please see any other information on materials and methods section
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of keratinocytes
- Justification for non-LLNA method:
- Skin sensitisers have been reported to induce genes that are regulated by the antioxidant response element (ARE). The KeratinoSens™ test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSens™ test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The method cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories lA and lB as defined by the UN GHS, for authourities implementing these two optional subcategories, nor to predict potency for safety assessment decisions. However, depending on the regulatory framework a positive result may be used on its own to classify a chemical int o UN GHS category 1.
Test material
- Reference substance name:
- Reaction mass of 2-methoxy-6-methylocta-1,5-diene and 2-methoxy-6-methylocta-2,5-diene
- EC Number:
- 950-170-3
- Molecular formula:
- C10H18O
- IUPAC Name:
- Reaction mass of 2-methoxy-6-methylocta-1,5-diene and 2-methoxy-6-methylocta-2,5-diene
Constituent 1
- Specific details on test material used for the study:
- Test Item Name: PG-RAW-0004
Supplier Code: PG-RAW-0004
Supplier batch/lot number: RDRW004-3
CAS number: 2109705-93-1; 2111193-86-1
Purity: 95.5%
Expiry Date: 01 Sep 19
Physical state: Clear liquid
Storage Conditions: Refrigerated
In vitro test system
- Details on the study design:
- Method of administration of test item:
Per plate, single application of 12 concentrations (2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 3.906, 1.953, 0.977 μM) of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing.
Method of administration of reference items:
Per plate, single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1% and single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate).
One well per plate is left empty (no cells).
Exposure times of test items and reference items:
Cells were incubated with the test or reference item for 48 ± 2h before endpoints measurements.
Number of repetitions:
Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence and 2 x 96-well plates for MTT. The validity of each repetition was assessed following acceptance criteria described.
Overview
Preliminary testing: Determination of the top concentration by solubility testing
Day 1: Seeding cells (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT); 10,000 cells per well, p14.
Day 2: 24h after seeding the test and control items were applied and plates were incubated at 37°C, 5% CO2, 2: ≥ 95% relative humidity for 48 ± 2h.
Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MTT testing (2plates)
Data Analysis
XCellR8 Forms F0056 A (version 01) and B (version 04): Data Analysis for KeratinoSensTM were used to analyse data. These forms are Microsoft Excel workbooks containing formulae to process the raw data as described in SOP L0057. The spreadsheets have been validated in-house (July 2017).
The following parameters were calculated in the KeratinoSensTM test method:
• the maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test item and positive control;
• the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5- fold threshold (i.e. 50% enhanced luciferase activity);
• For each concentration showing > 1.5-fold luciferase activity induction, statistical significance is calculated (e.g. by a two-tailed Student’s t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.
• The percentage of viability as compared to the Negative control
Results and discussion
- Positive control results:
- Positive Control (PC) (Cinnamic aldehyde) induced ≥1.5-fold in at least one concentration. The average induction of cinnamic aldehyde was 1.91
In vitro / in chemico
Results
- Run / experiment:
- mean
- Parameter:
- other: EC1.5
- Value:
- 11.5
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Other effects / acceptance of results:
- Solubility Assessment
The test concentrations of PG-RAW-0004 used in the KeratinoSensTM test were selected on the basis of solubility test carried out prior to the study:
Solubility of PG-RAW-0004 (MW: 154) in cell culture medium (confirmed up to 30.8mg/ml (200mM)); subsequent dilution in cell culture medium-1% DMSO giving a top concentration of 2000µM).
Discussion
The human skin sensitisation potential of PG-RAW-0004 was assessed using validated in vitro method: the KeratinoSensTM test to determine keratinocyte activation. The method was adapted to animal product-free conditions by XCellR8 and reference chemicals described in the guideline and in the performance standards were used to confirm the reliability, accuracy, sensitivity and specificity values. The adapted method showed full concordance with the Validated Reference Method (VRM) – the KeratinoSensTM standard protocol. We recently obtained clarification from the European Chemicals Agency (ECHA) that data using the adapted method may be used in REACH submissions, provided that the Performance Standards data, demonstrating equivalence with the VRM, is included in the dossier.
In this study, PG-RAW-0004 was classified as a sensitiser to human skin.
The sensitisation potential of PG-RAW-0004 was quantified by calculating 2 parameters known as the EC1.5 and the IMAX value. The meanings of these are as follows:
• The EC1.5 value means the Effective Concentration (EC) of test item that caused an induction of luciferase activity of greater than 1.5-fold over untreated controls. If at least one concentration induces luciferase activity to >1.5, then the product is classified as a skin sensitiser. (Note: this classification also requires the cell viability measured by MTT to be greater than 70%).
PG-RAW-0004 did cause luciferase induction >1.5 in all 3 repetitions. Therefore, PG-RAW-0004 was classified as a sensitiser.
• The IMax value is the maximum-fold induction observed within the concentration range tested. Although the KeratinoSensTM test is not validated to predict potency, the IMAX value can provide a useful tool for a very preliminary comparison of sensitisation potential between test items. As shown in Section 13.2, the maximum induction was observed at a test concentration of 2000µM, which showed an Imax value of 1904.107 in repetition 1; 35.506 at 2000µM in repetition 2; 2358.35 at 1000µM in repetition 3. For reference, during test validation, sensitising proficiency chemicals produced Imax values of up to 36-fold over untreated controls.
All of the formal acceptance criteria of the tests were met.
Any other information on results incl. tables
Determination of the skin sensitisation potential of PG-RAW-0004
Sensitisation Potential of the test item repetition 1
REP 1 |
Test item concentration (µM) |
|||||||||||
0.977 |
1.953 |
3.906 |
7.813 |
15.625 |
31.250 |
62.5 |
125 |
250 |
500 |
1000 |
2000 |
|
Mean of fold induction |
0.941 |
0.902 |
1.156 |
1.274 |
1.434 |
1.478 |
2.186 |
3.061 |
5.489 |
14.129 |
101.988 |
1904.107 |
SD |
0.039 |
0.045 |
0.049 |
0.085 |
0.150 |
0.273 |
0.091 |
0.219 |
0.945 |
0.920 |
9.893 |
104.463 |
Viability % |
118.13 |
108.40 |
96.08 |
96.65 |
94.49 |
95.13 |
107.09 |
112.29 |
130.72 |
136.18 |
157.23 |
140.53 |
Imax |
1904.107 at 2000µM |
|||||||||||
EC1.5 |
32.22µM |
|||||||||||
IC50 |
>2000µM as viability at 2000µM was 140.53% |
|||||||||||
IC30 |
>2000µM as viability at 2000µM was 140.53% |
Sensitisation Potential of the test item repetition 2
REP 2 |
Test item concentration (µM) |
|||||||||||
0.977 |
1.953 |
3.906 |
7.813 |
15.625 |
31.250 |
62.5 |
125 |
250 |
500 |
1000 |
2000 |
|
Mean of fold induction |
1.144 |
1.165 |
1.254 |
1.434 |
1.350 |
1.293 |
1.581 |
1.801 |
2.656 |
4.082 |
8.710 |
35.506 |
SD |
0.064 |
0.125 |
0.212 |
0.183 |
0.191 |
0.127 |
0.165 |
0.316 |
0.283 |
0.442 |
0.879 |
2.429 |
Viability % |
96.15 |
86.87 |
96.06 |
100.59 |
97.83 |
99.59 |
102.22 |
118.07 |
128.96 |
161.25 |
210.83 |
229.39 |
Imax |
35.506 at 2000µM |
|||||||||||
EC1.5 |
53.74µM |
|||||||||||
IC50 |
>2000µM as viability at 2000µM was 229.39% |
|||||||||||
IC30 |
>2000µM as viability at 2000µM was 229.39% |
Sensitisation Potential of the test item repetition 3
REP 3 |
Test item concentration (µM) |
|||||||||||
0.977 |
1.953 |
3.906 |
7.813 |
15.625 |
31.250 |
62.5 |
125 |
250 |
500 |
1000 |
2000 |
|
Mean of fold induction |
0.836 |
1.107 |
1.048 |
1.223 |
1.472 |
1.908 |
3.404 |
6.756 |
26.094 |
338.892 |
2358.350 |
0.010 |
SD |
0.040 |
0.202 |
0.073 |
0.081 |
0.115 |
0.098 |
0.303 |
0.713 |
1.935 |
35.363 |
123.870 |
0.003 |
Viability % |
95.90 |
88.13 |
90.31 |
103.20 |
100.58 |
103.66 |
110.91 |
134.53 |
152.64 |
164.49 |
158.82 |
0.43 |
Imax |
2358.35 at 1000µM |
|||||||||||
EC1.5 |
16.63µM |
|||||||||||
IC50 |
1687µM |
|||||||||||
IC30 |
1561µM |
Determination criteria for the skin sensitisation potential ofPG-RAW-0004 |
|||
|
REP1 |
REP2 |
REP3 |
Does at least one concentration ofTest Iteminduce luciferase activity>1.5-fold: |
Yes |
Yes |
Yes |
Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%: |
Yes |
Yes |
Yes |
Does EC1.5value occur at a concentration <1000µM (or <200µg/ml) |
Yes |
Yes |
Yes |
Does the test item induce the luciferase in a dose-dependent manner |
Yes |
Yes |
Yes |
Classification |
Sensitiser |
Sensitiser |
Sensitiser |
Assay Acceptane Criteria (Mean of the 3 repetitions)
Criteria |
Result |
Pass or Fail |
1-Positive Control (PC) (Cinnamic aldehyde) induction>1.5-fold in at least one concentration |
Yes |
Pass |
2-Average induction of PC at 32µM is [1.6-3.0] |
Yes (2.11) |
Pass |
3-EC1.5value is [6-39µM] |
Yes (11.50) |
Pass |
4-CV% of blank values < 20% |
Yes (13.06%) |
Pass |
Applicant's summary and conclusion
- Interpretation of results:
- study cannot be used for classification
- Conclusions:
- PG-RAW-0004 was classified as sensitiser
- Executive summary:
The human skin sensitisation potential of PG-RAW-0004 was assessed using the validated in vitro method, the KeratinoSensTMassay,adapted to fully animal-free by XCellR8,and validated in-house to determine keratinocyte activation.
After 48h exposure of cells with 12 concentrations of PG-RAW-0004,Luciferase measurements andMTT viability testing were performed.
PG-RAW-0004 was classified as sensitiser
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