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EC number: 203-854-4 | CAS number: 111-29-5
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Endpoint summary
Administrative data
Description of key information
In vitro Skin Sensitisation Test Battery with 1,5-pentanediol: no skin sensitizing potential
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2020-2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- ARE-Nrf2 luciferase LuSens test method
- Justification for non-LLNA method:
- .
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: B 2024 v. 23.09.2020
- Purity, including information on contaminants, isomers, etc.: 97.8 area-% (Analysis) - Details on the study design:
- The chemical and biological mechanisms associated with skin sensitisation are summarised in the form of an Adverse Outcome Pathway (AOP). This AOP includes four key events:
1) The molecular initiating event is the covalent binding of electrophilic substances to nucleophilic centres in skin proteins.
2) The inflammatory responses and gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways in the keratinocytes.
3) The activation of dendritic cells, typically assessed by expression of specific cell surfac markers, chemokines and cytokines.
4) The T-cell proliferation. This ARE-Nrf2 luciferase test method is proposed to address the second key event of the skin sensitisation AOP, namely keratinocytes activation. Skin sensitisers have been reported to induce genes that are regulated by the antioxidant response element(ARE). Small electrophilic substances such as skin sensitisers can act on the sensor protein Keap1(Kelch-like ECHassociated protein 1), by e.g. covalent modification of its cysteine residue, resulting in its dissociation from the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2). The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying enzymes.The test method is technically applicable to the testing of multiconstituent substances and mixtures. The test method is applicable to test items which are soluble or could be formulated as a homogeneous suspension/dispersion either in DMSO, water or culture medium.A LuSens prediction should be considered in the framework of a Defined Approach or of an IATA and in accordance with the provision paragraph 4 and paragraphs 7 and 8 of the OECD 442D general introduction.The ARE-Nrf2 luciferase test (LuSens) can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), human cell line activation test method (h-CLAT)) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.The technical proficiency of the LuSens with the OECD 442D guideline recommended proficiency substances was demonstrated. - Vehicle / solvent control:
- DMSO
- Negative control:
- DL-Lactic acid
- Positive control:
- EGDMA (120 M) [442D]
- Key result
- Group:
- test chemical
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Remarks:
- 1,5-Pentanediol did not activate the LuSens cells up to a concentration of 2000 µM under the test conditions of this study.
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In conclusion, the test item 1,5-Pentanediol did not activate the LuSens cells up to a concentration of 2000 µM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).
- Executive summary:
This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization AOP) of 1,5-Pentanediol. Dose calculation was adjusted to purity to test a final maximal concentration of 2000 µM of the test item. Due to adaptation of the purity of 97.8 area-% instead of 97.96 area-% after experimental completion at request of the sponsor, the adjustment to purity actually carried out resulted in a maximum concentration <2000 µM. Since the deviation occurred in the calculation was in the range <1%, the undercutting of the guideline defined maximum concentration is evaluated as biologically irrelevant. In the cytotoxicity test, cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (2000 µM). Due to the lack of cytotoxicity, a CV75 value could not be calculated. In this case, the OECD 442D guideline recommend to test a test item concentration of 2000 µM in the main experiments. Five further test item dilutions were prepared by serial dilution with a dilution factor of 1.2. The test item was tested in 4 independent main experiments. The following concentrations of the test item were tested in the main experiments: 804, 965, 1157, 1389, 1667, 2000 µM In the first experiment two concentrations (804 and 1667 µM) showed a luciferase induction ≥ 1.5 fold compared to the solvent control. Since, theses concentrations were not consecutive, the first experiment is considered negative. In the second experiment only the highest tested concentration of 2000 µM was < 1.5 fold. All other concentrations showed a luciferase induction in a range of 5.19 to 5.75 fold. Therefore the second experiment is considered positive. In the third experiment all tested concentrations showed luciferase induction < 1.5 fold and is considered negative. For better interpretation of the date, a fourth experiment was performed. The fourth experiment confirmed the negative outcome of the first and third experiment. After treatment with the test item for 48 ± 1 hours the luciferase induction is < 1.5 fold compared to the solvent control in three of four experiment. Therefore the LuSens prediction is considered negative.
The acceptance criteria were met:
The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 1: 7.33; ME 2: 6.50; ME 3: 6.25; ME 4: 4.31) and statistically significant. The positive control had a relative cell viability ≥ 70% as compared to the solvent control (ME 1: 111.53%; ME 2: 137.01%; ME 3: 126.21; ME 4: 92.19). The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells was < 1.5 fold as compared to the average solvent control (ME 1: 1.11; ME 2: 1.21; ME 3: 1.12; ME 4: 1.01). The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20% in each main experiment (ME 1: 7.5%; ME 2: 10.2%; ME 3: 6.5; ME 4: 7.3). At least three test item concentrations had a cell viability of at least 70% relative to the solvent controls. The maximum concentration of 2000 μM has been tested.- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2020-2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 442E (In Vitro Skin Sensitisation: Activation of Dentritic Cells)
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- human Cell Line Activation Test (h-CLAT)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: B 2024 v. 23.09.2020
- Purity, including information on contaminants, isomers, etc.: 97.8 area-% (Analysis) - Details of test system:
- THP-1 cell line [442E]
- Details on the study design:
- The h-CLA T is a dendritic cell activation test to predict skin sensitizing potential. The test is performed using the human pro-monocytic cell line THP-1 as surrogate for dendritic cells. As readout, the change in the expression of the cell membrane markers CD 54 and CD 86 measured by flowcytometry after 24 hours of test substance exposure. A test substance is predicted to be a skin sensitizer when marker expression exceeds the th reshold in relation to vehicle control.The test item concentrations investigated in the main experiment (h-CLAT) were determined with two cytotoxicity tests. The tests were performed with independent cell cultures (cells are collected from different culture flasks). The test item was prepared separately for each run.The test item dilutions were prepared freshly before each experiment. Each volume (500 µL) of the dilutions of the test item and culture medium was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. All dose groups were tested in one replicate for each cytotoxicity test. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations. Each concentration of the test item, culture medium and solvent control were prepared for the 7-AAD staining.
Each test item-treated and not test item-treated cells were collected in sample tubes, centrifuged (approx. 250 × g, 5 min), washed twice (2 - 8 °C) with 2 mL FACS buffer (PBS with 0.1% (w/v) BSA) and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added in each sample tube.
Flow Cytometry Acquisition (Cytotoxicity Test)
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.Thecytotoxicity was analysed by flow cytometry using the software Cellquest Pro 6.0. The 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal. On the day of the cytotoxicity or main experiment (h-CLAT) directly before the treatment of the cells, a volume of 500 µL with a cell density of 1.8 - 2 × 106 THP-1 cells/mL was seeded in each corresponding well of a 24-well flat bottom plate.
Preparation for CD54 and/or CD86 expression measurements/cell staining:
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
For CD86/CD54 expression measurement, each test item is tested in at least two independent
runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE (see chapter 5.6.7.1).Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline) - Vehicle / solvent control:
- DMSO
- Positive control:
- dinitrochlorobenzene (DNCB) [442E]
- Key result
- Group:
- test chemical
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Remarks:
- 1,5-Pentanediol did not activate THP-1 cells up to a concentration of 5000 µg/mL under the test conditions of this study.
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item 1,5-Pentanediol with a log Pow of -0.49 did not activate THP-1 cells up to a
concentration of 5000 µg/mL under the test conditions of this study. Therefore, the test item
is considered negative for the third key event of the skin sensitisation Adverse Outcome
Pathway (AOP). - Executive summary:
This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of 1,5-Pentanediol dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 (luciferase test method)) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals. In the first cytotoxicity test a technical error occurred. Therefore the test was cancelled and an additional cytotoxicity test was performed. Cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (5000 µg/mL). Due to the lack of cytotoxicity, a CV75 value could not be calculated. Therefore, the OECD 442E guideline recommended maximal to be tested test item concentration (5000 µg/mL) was used for the h-CLAT runs. The following concentrations of the test item were tested in the main experiments (h-CLAT): 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 µg/mL. The test item with a log Pow of -0.49 was tested in 2 independent runs. The relative fluorescence intensity (RFI) of CD86 and/or CD54 was not equal or greater than 150% and 200%, respectively at any dose in both runs. Therefore, the h-CLAT prediction is considered negative for the test item in this h-CLAT. In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was > 50%.
In conclusion, the test item 1,5-Pentanediol with a log Pow of -0.49 did not activate THP-1 cells up to a concentration of 5000 µg/mL under the test conditions of this study. Therefore,the test item is considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2020-2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: B 2024 v. 23.09.2020
- Purity, including information on contaminants, isomers, etc.: 97.96 area-% (Analysis)* - Details of test system:
- cysteine peptide, (Ac-RFAACAA-COOH)
- lysine peptide (Ac-RFAAKAACOOH)
- Details on the study design:
- Skin sensitisation (In chemico test system) - Details on study design:
Due to the complexity of the skin sensitization process a single in vitro assay is not sufficient to adequately assess this toxicological endpoint. Therefore, a combination of several methods addressing key events of the sensitization process: protein reactivity (DPRA), activation of keratinocytes (LuSens or KeratinoSens) and activation of dendritic cells (MUSST or h-CLAT) were used (test battery).
Direct Peptide Reactivity Assay (DPRA): Chemical reactivity has been shown to be well associated with allergenic potency (Gerberick et al., 2007) and has been described as the molecular initiating event in the OECD adverse outcome pathway (OECD Publication No.168; ENV/JM/MONO(2012)10). Within this context measuring the amount of proteins with nucleophilic side chains such as cysteine or lysine residues after incubation with putative allergens serves as surrogate markers. In the DPRA the reactivity of a test item (=test substance) towards synthetic cysteine (C)- or lysine (K)-containing peptides is evaluated. For this purpose, the test substance is incubated with synthetic peptides for 24 hours and the remaining non-depleted peptide concentration is determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm.
The peptide depletion of test-substance incubated samples is compared to the peptide depletion of the NC samples and expressed as relative peptide depletion.
The test substance was dissolved in a suitable vehicle. Three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (=NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method. - Vehicle / solvent:
- acetonitrile
- Positive control:
- other: EGDMA
- Positive control results:
- Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), prepared as a 50 mM solution in acetonitrile.
- Key result
- Group:
- test chemical
- Parameter:
- other: Mean peptide depletions of Cysteine, Lysine and both peptides mean of both depletions
- Value:
- 0.22 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Group:
- test chemical
- Parameter:
- mean cystein depletion
- Value:
- 0.23 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Group:
- test chemical
- Parameter:
- mean lysine depletion
- Value:
- 0.21 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that 1,5-Pentanediol shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.
- Executive summary:
he reactivity of 1,5-Pentanediol towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at 100 mM in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, in order to detect possible interference of the test substance with the peptides, a co-elution control was performed and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm.
The following results were obtained in the DPRA:
The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. No co-elution of test substance and peptides was present. The mean C-peptide depletion, caused by the test substance was determined to be 0.23%. The mean K-peptide depletion, caused by the test substance was determined to be 0.21%. Thus, the mean peptide depletion was calculated to be 0.22%.
Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that 1,5-Pentanediol shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.
Referenceopen allclose all
Results and historic control data are listed in the annex under "attached background material"
Results and historical control data are listed in the annex under "attached background material"
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
Testing 1,5-Pentanediol in three different non-animal methods addressing different key events of the skin sensitization Adverse Outcome Pathway resulted in three negative results in the h-CLAT, DPRA and LuSens. The three tests generally used in the "2 out of 3" DEFINED APPROACH assess protein binding in chemico (DPRA), triggering an antioxidant response in keratinocytes (LuSens) and activation of dendritic cells (h-CLAT). Of note, the molecular initiating event of protein binding also occurs in the cell-based assays (LuSens, h-CLAT) and is not solely detected via the DPRA.
Based on the results, 1,5-Pentanediol is not peptide reactive, does not activate keratinocytes and does not activate dendritic cells. In conclusion, 1,5-Pentanediol is predicted not to be a skin sensitizer.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
Justification for classification or non-classification
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218. As a result the substance is considered to be not classified as skin sensitising.
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.
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