1-hydroxyoctan-2-one

Endpoint summary

Description of key information

For determination regarding the dermal sensitization potential of 1 -hydroxyoctan-2 -one, a combination of in chemico and in vitro tests were conducted (OECD 422C, OEC 422D and OECD 422E) and combined with in silico QSAR analysis. Each of the three in vitro tests are considered to be reliable and, apart from the DPRA (sensitiser with low reactivity), resulted in no evidence of skin sensitization. Using in silico approaches with the OECD QSAR toolbox software the major tautomeric form of the substance (ketone) was determined to exhibit no flags for skin sensitisation for KE1 (covalent binding to skin proteins). The substance is within the applicability domain for the model.

According to CLP the in vitro / in chemico tests are not regarded as stand alone tests and the result from such a test should be used together with other data in an overall weight of evidence assessment.

There are no in vivo animal data (LLNA or non-LLNA) available to make decisions about classification.

Based on a weight of evidence (WoE) approach using all available data (human data, in chemico / in vitro data and in sillico/QSAR), 1 -hydroxyoctan-2 -one is not classified as a skin sensitiser

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This is a Klimisch 1 OECD 442C guideline study conducted on the registered substance 1-hydroxyoctan-2-one in accordance GLP

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)

Justification for non-LLNA method:

OECD TG 442C cites the DPRA model as a validated method for skin sensitisation testing in the context of an integrated approach to testing and assessment.

The DPRA has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-led validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid to be used as part of an Integrated Approach to Testing and Assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Specific details on test material used for the study:

Batch 1081-67E

Details on the study design:

The DPRA is proposed to address the molecular initiating event of the skin sensitisation Adverse Outcome Pathway (AOP), namely protein reactivity, by quantifying the reactivity of test and reference items towards model synthetic peptides containing either Lysine or Cysteine. Cysteine and Lysine percent peptide depletion values are then used to categorise a substance in one of four classes of reactivity for supporting the discrimination between skin sensitisers and non-sensitisers.

Test items were incubated for 24hrs (±2hrs) at 25 ±2.5˚C in solution at 100mM in combination with either Cysteine or Lysine containing peptides and then run on an HPLC system (20-minute run-time) using gradient elution and UV detection at 220nm to measure peptide concentration. Test items were compared to reference controls containing the test item solvent in combination with either Cysteine or Lysine peptide in order to determine the relative percent peptide depletion. Relative percent peptide depletion values were used in a prediction model that assigns test items to one of four reactivity classes.

Positive control results:

Acceptance Criteria

Acceptance criteria for all controls and the test item were met in both runs with the exception of the Standard Curve r2 value and the RefA and RefC mean peptide concentrations for the Cysteine run (highlighted in orange below). However, the Standard Curve (r2=0.975) only narrowly missed the r2 value acceptance criterion and therefore was considered suitable for use. The marginally decreased RefA (0.393mM) and RefC (0.413mM) concentrations were not deemed to have affected the result of the testing as the test item was prepared using the same peptide and solvent and therefore is comparable to the RefC control. In addition, the test item caused little/no Cysteine peptide depletion. These controls were considered acceptable for the purposes of the study based upon the overall result and the performance of all other controls.

Data exclusion

Cysteine peptide RefB4 and RefC2 were excluded in line with guidance in XCellR8 SOP L0096 regarding data exclusion. They were excluded as they were clear outliers that had pushed the Peak Area %CV beyond the acceptance range. After removal of outliers, acceptance criteria for Peak Area %CV were met for both RefB and RefC (11.850 and 6.149 respectively).

Key result

Run / experiment:

other: 1

Parameter:

other: mean % peptide depletion (Cys+Lys)

Value:

8.989

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Sensitiser with Low Reactivity

Run / experiment:

other: 1

Parameter:

other: %cysteine peptide depletion

Value:

1.449

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Run / experiment:

other: 1

Parameter:

other: % lysine depletion

Value:

16.529

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Name	Test Item ID	% Cysteine Peptide Depletion	% Lysine Peptide Depletion	Mean % Peptide Depletion (Cys + Lys)	DPRA Prediction	DPRA Reactivity Class
EXPINN PC17032	TA1	1.449	16.529	8.989	Sensitiser	Low Reactivity

Interpretation of results:

GHS criteria not met

Conclusions:

1 -hydroxyoctan-2 -one was classified as a Sensitiser with Low Reactivity as per the prediction model.

Executive summary:

In this study, the skin sensitisation potential of 1 -hydroxyoctan-2 -one was assessed using theIn Chemico Direct Peptide Reactivity Assay (DPRA) method according to OECD Test Guideline 442C. After a 24h incubation with both Cysteine and Lysine containing peptides, the percent peptide depletion was measured by High Performance Liquid Chromatography (HPLC).

 

The test item produced 8.989% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test item was classified as a Sensitiser with Low Reactivity. A single HPLC analysis for both the Cysteine and the Lysine peptide was considered sufficient for the test item as the result was unequivocal for each of the peptides individually.Even though the result fell in the region of 3-10% depletion for the Cysteine 1:10 / Lysine 1:50 prediction model when results for both peptides were combined, i.e. close to the threshold (6.38%) use to discriminate between sensitisers and non-sensitisers, it was not considered necessary to repeat the runs due to the nature of the reactivity with each peptide individually. The reasons for this and the results are summarised below:

The result with the Cysteine peptide indicated little to no reactivity of the test item with the peptide across all replicates

The result with the Lysine peptide showed consistent reactivity with the peptide across all replicates with very little variation in the data as evidenced by the standard deviation and %CV values

The final mean % peptide depletion observed using this model was 8.989%. Therefore, 1 -hydroxyoctan-2 -one was classified as a Sensitiser with Low Reactivity as per the prediction model.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This is a Klimisch 1 OECD 442D guideline study conducted on the registered substance 1-hydroxyoctan-2-one in accordance GLP

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:

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 KeratinoSensTM 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 KeratinoSensTM 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 1A and 1B as defined by the UN GHS, for authorities 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 into UN GHS category 1.

Specific details on test material used for the study:

Batch 1081-67-E

Details on the study design:

The KeratinoSensTM cell line (test system) is an immortalized adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.

A single application of 12 concentrations 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 using a cytotoxicity test with human fibroblasts in the preliminary test.

A 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%.

A single application of culture medium with 1% DMSO was applied as the negative control.

The cells were incubated for 48 ± 2h before endpoints measurements

Three runs are required. Each run includes 3 x 96-well plates for luminescence and 1 x 96-well plate for MTT. The validity of each run was assessed following acceptance criteria described in the study report.

Positive control results:

1. Positive Control (PC) (Cinnamic aldehyde) induction >1.5-fold in at least one concentration: PASS
2. Average induction of PC at 32μM is [1.6-3.0]: PASS
3. EC1.5 value is [16-32μM]: FAIL (<8uM)*

*Note: Criterion 3 failed. However, Criterion 2 passed an cinnamic aldehyde showed a dose-response curve and therefore the test was considered valid.

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5

Remarks:

effective concentration of test item causing induction of luciferase activity >1.5-fold over untreated controls

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

None of the concentrations of the test item caused luciferase induction >1.5. Therefore, no EC1.5 value can be calculated.

Run / experiment:

other: 1

Parameter:

other: Imax

Remarks:

maximum fold-induction observed within conc range tested

Value:

1.127

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Basic cytotoxicity to cells in vitro in the absence of the protective skin barrier. (Human dermal fibroblasts in animal product-free culture). The top non-cytotoxic concentration of the test item to human dermal fibroblasts was 250 μg/ml. A top test concentration of 400 μg/ml was selected for this study, to ensure detection of sensitising potential at the maximum possible concentration.

Solubility of test item in cell culture medium (confirmed up to 200mg/ml in DMSO; subsequent dilution in cell culture medium giving a top concentration of 2000μg/ml).

	Test Item Concentration (μg/ml)
	0.195	0.391	0.781	1.563	3.125	6.25	12.5	25	50	100	200	400
Mean of fold-induction	1.023	0.999	1.127	1.104	1.105	0.975	1.036	0.992	0.797	0.602	0.388	0.226
SD	0.169	0.125	0.142	0.099	0.186	0.188	0.143	0.116	0.101	0.068	0.042	0.041
Viability	99.92	98.05	104.55	103.29	100.23	91.49	100.75	100.2	107.14	114.75	122.26	112.71
IMAX	1.127 at 0.781μg/ml
EC1.5	Not Determined

Determination criteria for skin sensitisation potential of Test Item
Does at least one concentration of Test Item induce luciferase activity >1.5-fold:											NO
Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%:											N/A
Does EC1.5 value occur at a concentration <1000μM (or <200μg/ml)											N/A
Does the test item induce the luciferase in a dose-dependent manner											N/A
Classification											Non-Sensitiser

Interpretation of results:

GHS criteria not met

Conclusions:

The test item 1 -hydroxyoctan-2 -one was not classified as a skin sensitiser in this study

Executive summary:

The human skin sensitisation potential of 1 -hydroxyoctan-2 -one was assessed using the validated in vitro method, the KeratinoSensTM assay, adapted to fully animal-free by XCellR8, and validated in-house to determine keratinocyte activation.

After 48h exposure of cells with 12 concentrations of 1 -hydroxyoctan-2 -one, Luciferase measurements and MTT viability testing were performed. The sensitisation potential of EXPINN PC17032 was quantified by calculating 2 parameters known as the EC_1.5 and the I_MAX value.

None of the concentrations of 1 -hydroxyoctan-2 -one caused luciferase induction >1.5. Therefore, no EC1.5 value can be calculated. On the basis of this result the substance is classified as a non-sensitiser.

The maximum induction (I_MAX) was observed at a test concentration of 0.781μg/ml, which yielded a value of 1.127. For reference, during test validation, sensitising proficiency chemicals produced Imax values of up to 36-fold over untreated controls.

The test item 1 -hydroxyoctan-2 -one was not classified as a skin sensitiser in this study.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May-June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This is a Klimisch 1 OECD 442E guideline study conducted on the registered substance 1-hydroxyoctan-2-one in accordance GLP

Qualifier:

according to guideline

Guideline:

other: OECD Test Guideline 442E

Version / remarks:

In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT) [Version July 2016]

Principles of method if other than guideline:

Skin sensitisers have been reported to induce the expression of cell membrane markers associated with Dendritic Cell (DC) activation. The h-CLAT method is an in vitro assay that quantifies these changes in cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells (a cell line that mimics DCs), following 24-hour exposure to the test item. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface maker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

OECD TG 442E cites the h-CLAT model using THP-1 cells as a validated method for skin sensitisation testing in the context of an integrated approach to testing and assessment (IATA)

The h-CLAT has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-lead validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid to be used as part of an Integrated Approach to Testing and Assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Specific details on test material used for the study:

Batch 1081-67E

Details on the study design:

Solubility was first determined for the test item using either culture medium (RPMI 1640) or DMSO. Note that for this method, test items with a Log Kow (octanol/water partition coefficient) of up to 3.5 have been tested successfully. However, test items with a Log Kow of greater than 3.5 can still be tested at lower soluble concentrations. In such a case, a negative result (non-sensitiser) should be considered inconclusive, whereas a positive result (sensitiser) could still be considered valid.

THP-1 cells were pre-cultured for either 48 or 72hrs. Following this, the cells were dosed with the test item over an 8 dose range and incubated for 24 ±0.5hrs. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The dose of test item that yields 75% cell viability (CV75) was calculated and taken forward for the next stage of testing. This dose finding assay was carried out over two independent runs.

THP-1 cells were pre-cultured again for either 48 or 72hrs. Once the CV75 was determined, a narrower dilution series based around the CV75 value was produced for the test item. This dilution range was used to dose the cells again for 24 ±0.5hrs. The cells were then washed and stained with propidium iodide and also with antibodies that detect CD54 and CD86 expression as well as a negative control antibody. This allowed for discrimination of live/dead cells and also changes in CD54 and CD86 marker expression by flow cytometry.

Positive control results:

In the positive control (Nickel Sulphate), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be greater than 50%.Results below:

Run 1
RFI = 162; Viability = 88.32 (CD86)
RFI = 276; Viability = 87.18 (CD54)

Run 2
RFI = 169; Viability = 91.22 (CD86)
RFI = 235; Viability = 90.85 (CD54)

Hence the positive control performance met all acceptance criteria

Key result

Run / experiment:

other: 1

Parameter:

other: Relative Fluorescence Intensity (RFI)%

Remarks:

RFI CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative activation of dendritic cells positive according to OECD 442E

Remarks:

Viability >50%

Key result

Run / experiment:

other: 1

Parameter:

other: Relative Fluorescence Intensity (RFI)%

Remarks:

RFI CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative activation of dendritic cells positive according to OECD 442E

Remarks:

Viability >50%

Other effects / acceptance of results:

Assay acceptance criteria

Run acceptance criteria
 
•                   Cell viability of medium and DMSO controls should be greater than 90%
•                   In the positive control (Nickel Sulphate; 100µg/ml), RFI values of both CD86 and CD54 should be over the positive criteria (CD86≥150 and CD54≥200) and cell viability should be > 50%
•                   In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86≥150 and CD54≥200)
•                   For both medium and DMSO controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%

Data acceptance criteria
 
•                   For a test item resulting in a negative value (i.e. Non-Sensitiser), the cell viability at the 1.2 x CV75 value should be less than 90%
•                   For a test item resulting in a positive value (i.e. Sensitiser), a cell viability at the 1.2 x CV75 value of more than 90% is considered acceptable
•                   When test items in RPMI are tested at 5mg/ml or test items in DMSO are tested at 1mg/ml or the highest soluble concentration is used as the maximal dose instead of the CV75 based dose, the data for the test item are accepted regardless of the cell viability
•                   Cell viability of at least 4 doses in each assay should be > 50%.
 
Abnormal Values

•            RFI values cannot be less than zero. Such values should be excluded from the prediction.

Prediction Model
 
Number of runs required for prediction
 
At least two independent runs need to be performed to derive a final prediction. If a more precisely derived EC value is required, three independent runs should be performed. If 2 runs are carried out the higher of the EC values is taken as the final value. If 3 runs are carried out, then the median EC value is taken as the final result. Up to 6 total runs, meeting the “Run acceptance criteria”, may be performed in order to reach a conclusion for each test item. The six runs may include runs for which the “Data acceptance criteria” are not met for this test item e.g. Run 1: invalid, 2: valid, 3: invalid, 4: invalid, 5: valid and 6: invalid. If no prediction can be made after the sixth run the result is inconclusive and the test item is to be classified accordingly.

 Prediction Model Criteria
 
If the RFI of CD86 is equal to or greater than 150% at any tested dose (≥ 50% cell viability) in at least 2 independent runs, AND/OR the RFI of CD54 is equal to or greater than 200% at any tested dose (≥ 50% cell viability) in at least 2 independent runs, the test item prediction is considered Positive (Sensitiser). Otherwise the result is considered as Negative (Non-Sensitiser). In case the first 2 runs are not concordant, a third run needs to be performed and the final prediction will be based on the mode of the conclusions from the three individual runs (i.e. 2 out of 3).

Maximal Doses and the prediction model
 
When test items in RPMI are tested at 5mg/ml or test items in DMSO are tested at 1mg/ml or the highest soluble concentration is used as the maximal dose instead of the CV75 based dose and the data for the test item does not meet the positive criteria for the prediction model without affecting the cytotoxicity at all tested doses, the test item prediction should be considered as negative.
 

The CV75 dose informs the dosing range selected for the CD54/86 expression assay. The top dose for the CD54/86 expression assay (main test) is 1.2 x CV75 which was equal to 1.2mg/ml (1200µg/ml).The following tables show the expression of CD54 and CD86 against test item dose with concurrent cytotoxicity measurement:

Run 1 (Valid): Result = Non-Sensitiser

Test Item Dose (µg/ml)	Cell Viability (%)			Average Cell Viability (%)	CD54 RFI	CD86 RFI
	Isotype	CD54	CD86
1200.00	77.18	78.95	79.90	78.68	78	45
1000.00	81.31	84.20	85.48	83.66	70	63
833.33	88.80	89.64	89.98	89.47	95	105
694.44	95.33	95.60	95.50	95.48	89	108
578.70	94.72	94.21	95.55	94.83	107	135
482.25	94.56	94.67	94.72	94.65	95	103
401.88	94.55	94.41	95.10	94.69	97	109
334.90	96.37	96.07	96.37	96.27	83	103

Run 2 (Valid): Result = Non-Sensitiser

Test Item Dose (µg/ml)	Cell Viability (%)			Average Cell Viability (%)	CD54 RFI	CD86 RFI
	Isotype	CD54	CD86
1200.00	78.36	75.81	74.55	76.24	152	70
1000.00	77.60	80.80	80.43	79.61	152	54
833.33	74.42	74.68	77.59	75.56	123	78
694.44	78.69	79.33	83.30	80.44	87	74
578.70	94.34	92.82	94.80	93.99	100	124
482.25	89.43	88.43	89.05	88.97	127	71
401.88	92.85	92.48	94.11	93.15	108	74
334.90	96.67	97.27	96.67	96.87	103	102

As can be seen from the data, the expression of CD54 as measured by the RFI did not cross the threshold (RFI ≥200) at any of the doses tested. The expression of CD86 as measured by the RFI did not cross the threshold (RFI ≥150) at any of the doses tested. As the CD54/CD86 expression did not cross the threshold the test item is classified as a Non-Sensitiser. Cell viability did not fall below 50% at any of the test item concentrations and therefore the result is deemed to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

1-hydroxyoctan-2-one was classified as a non-sensitiser using the prediction model in accordance with OECD TG 442E

Executive summary:

The skin sensitising potential of the test substance, 1 -hydroxyoctan-2 -one, was determined via the in vitro Human Cell Line Activation Test (h-CLAT) that evaluates that ability of the substance to induce the expression of cell membrane markers (CD86 and CD54) and thus activate dendritic cells. The study was conducted following the OECD Guideline for Testing of Chemicals TG. 442E, adopted July 2016 (‘In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)’)

For the test item 1 -hydroxyoctan-2 -one the maximum possible dose in DMSO for the CV75 assay (1mg/ml; (1000µg/ml)) did not reduce cell viability to below 75%, therefore the CV75 was taken as >1mg/ml (>1000µg/ml).

The sensitisation threshold for CD54 and CD86 expression in the main test was not crossed in either of the runs using a maximal dose of 1.2mg/ml (1200µg/ml) and therefore the test item was classified as a Non-Sensitiser as per the prediction model.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the negative outcome in both KeratinoSens assay (OECD 442D) and h-CLAT assay (OECD 442E), combined with in silico predictions using the OECD QSAR toolbox the substance is not predicted to be a skin sensitiser. The substance does not meet the criteria for classification.