1-methoxyhexane

Endpoint summary

Description of key information

Skin sensitization (DPRA, OECD 442C): not sensitizing

Skin sensitization (KeratinoSens, OECD 442E): not sensitizing

Skin sensitization (h-CLAT, OECD 442D): sensitizing

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:

key study

Study period:

3 - 11 March 2017

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

insert date

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Synthetic peptide containing cysteine: Ac-RFAACAA-COOH, lot number 1556171, purity 95% (by HPLC), supplied by AnaSpec, stored frozen (-10°C to -30°C).
Synthetic peptide containing lysine: AC-RFAAKAA-COOH, lot number 1556172, purity 94% (by HPLC), suppoed by AnaSpec,stored frozen (-10°C to -30°C).

Positive control: cinnamic aldehyde, purity > 95%, was prepared at a concentration of 100 mM in acetonitrile

Preparation of peptide stock solutions:
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (for cysteine, 100 mM phosphate buffer pH 7.5, for lysine 100 mM ammonium acetate buffer pH 10.2).

Preparation of peptide calibration standards:
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Reference (Stability) Controls and Precision Controls:
Reference (stability) controls and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile. These were injected throughout the analytical run to confirm consistency of peptide response throughout each analytical run.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls:
A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials. Cysteine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in more acetonitrile and cysteine peptide stock solution. The final sample concentration was 5 mM of the test substance, 0.5 mM cysteine.
In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 5 mM with 0.5 mM cysteine.
The co-elution control sample contained 5 mM of the test substance in phosphate buffer solution. An additional control sample of 5 mM of Diola BHT in acetonitrile was also prepared so as to positively identify peak(s) of the test item in the co-elution control ensuring that the test item had not evaporated off during incubation and injection of the samples on the HPLC.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls:
A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials. Lysine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM
test substance solution in lysine peptide stock solution. The final sample concentration was 25 mM of the test substance, 0.5 mM lysine.
In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 25 mM with 0.5 mM lysine. The co-elution control sample contained 25 mM of the test substance in ammonium acetate buffer solution. An additional control sample of 25 mM of Diola BHT in acetonitrile was also prepared so as to positively identify peak(s) of the test item in the co-elution control ensuring that the test item had not evaporated off during incubation and injection of the samples on the HPLC.

Incubation:
The appearance of the test substance, positive control samples and co-elution controls in the HPLC vials was documented following preparation with the vials then placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis:
The concentration of both the cysteine and lysine peptides in the presence of the test substance and the associated positive controls were quantified by HPLC using UV detection.
Equipment: HPLC Waters Alliance 2695 separation module an d2487 dual wavelength detector.
Column: Agilent Zorbax SB C18, 3.5 µm, 100 × 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30 °C
Sample temperature: 25 °C
Mobile phase A: 0.1% trifluoroacetic acid in water
Mobile phase B: 0.085% trifluoroacetic acid in acetonitrile
Flow rate: 0.35 mL/minute
Detector wavelength: UV, 220 nm
Injection volume: 2 μL
Run time: 30 minutes
Approximate retention time (cysteine): 11 minutes
Approximate retention time (lysine): 7 minutes

Calculations:
The peak area response for each peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:

% peptide depletion = 100 - [(Peptide peak area in replicate depletion samples x 100) / (Mean peptide peak area of reference (stability) control samples)]

Positive control results:

69.9% depletion (SD 0.21%, n = 3) and 57.9% depletion (SD 1.26%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.

Key result

Run / experiment:

other: 1

Parameter:

other: cysteine depletion, %

Value:

-1.31

Vehicle controls validity:

valid

Remarks:

stability and precision controls

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: lysine depletion, %

Value:

-0.821

Vehicle controls validity:

valid

Remarks:

stability and precision controls

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for reference (stability) controls and precision controls of both peptides: yes, CV 0.97%, n = 6 and CV 0.26%, n = 6, for cysteine and lysine, respectively, at 0.50 and 0.51 mM).
- Acceptance criteria met for positive control: yes, 69.9% depletion (SD 0.21%, n=3) and 57.9% depletion (SD 1.26%, n=3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.
- Acceptance criteria met for variability between replicate measurements: yes, SD 0.81% (n=3) and SD 0.74% (n=3), respectively, for cysteine and lysine depletion by the test item.

TEST SUBSTANCE RESULTS:
Mean depletion of –1.31% and -0.821% was observed for the test substance with cysteine and lysine peptides, respectively. With the test substance not reacting with the cysteine nor lysine peptide it is classed as “no to minimal”, hence the DPRA prediction is negative.

Interpretation of results:

other: DPRA was negative

Conclusions:

It can be concluded that this DPRA test is valid, and that the test substance was negative in the DPRA and is classified in the “no or minimal reactivity class” when using the Cysteine / Lysine prediction model.

Executive summary:

In a GLP-compliant OECD guideline 442C study, Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity of the substance by protein depletion being one part of its sensitisation mechanism. In the cysteine and lysine reactivity assay all analytical acceptance criteria of the test were met. The test substance caused -1.31% (SD 0.81%) and -0.821% (SD 0.74%) cysteine and lysine peptide depletion, respectively. These results are categorised as “no to minimal reactivity” based on the DPRA prediction model and is thus considered to be negative in the DPRA.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 February - 30 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 442E: In vitro skin sensitization: human Cell Line Activation Test (h-CLAT)

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 14 September 2015

Type of study:

other: human Cell Line Activation Test (h-CLAT)

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
Solvent: DMSO (final concentration 0.2% in culture medium, also used as a solvent for positive control)
Positive control: DNCB in DMSO diluted with culture medium (2 and 3 μg DNCB/mL)

Cell line: THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers. The cell density did not exceed 1 × 10^6 cells/mL. The passage numbers of the used THP-1 cells was 13 in both XTT assays and 19 and 24 in the h CLAT for runs 1 and 2, respectively.
Culture medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) was used to culture the cells during the assay.
Preparation and seeding of THP-1 cells: On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 μL with a cell density of 0.9 - 1 × 10^6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate. For the main experiment (h-CLAT) 0.9- 1 × 10^6 cells/well in a volume of 500 μL was seeded in a 24-well plate before the treatment.

Dose finding assay: The doses investigated in the main experiment (h-CLAT) were determined with two XTT tests, instead of flow cytometry recommended by the guideline.The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl)-(3,4-tetrazolium)–bis-(4–methoxy–6-nitro)-benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. Two independent cytotoxicity experiments were performed with different cell cultures days to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT). CV75 is defined as the concentration of toxicant required to reduce the relative absorbance to 75% of the solvent control and is calculated as:
CV75 = Conc.>75 - [(Conc.>75 - Conc.<75) x (%>75 - 75)]/(%>75 - %<75), where:
a) Conc.>75 = maximal measured concentration with the % of solvent control > 75%
b) Conc.<75 = minimal measured concentration with the % of solvent control < 75%
c) %>75 = relative absorpbance at a) in %
d) %<75 = relative absorpbance at b) in %
Test item preparation: immediately prior to start the substance was dissolved in culture medium. The maximum concentration of test item was 5000 μg/mL in culture medium, as tested by a solubility test. For the XTT test (dose finding assay) eight concentrations of the test item were analysed. Therefore, dilutions were prepared by 1:2 serial dilutions from 5000 μg/mL in culture medium.
XTT Labelling and Measurement: At the end of the incubation period, 50 µL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Acceptability criteria of XTT assay:
The XTT test is considered to be acceptable if it meets the following criteria:
• mean absorbance of the medium control is ≥ 0.5
• mean viability of the solvent control is ≥ 90% in comparison to the medium control

Main test:
The test item was tested in two independent runs. The second run was cancelled and repeated due to a technical error.
Test item preparation: For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. The following concentrations of the test item (solved in culture medium) were tested in the main experiment (h-CLAT): 727, 872, 1046, 1256, 1507, 1808, 2170 and 2604 μg/mL.
Treatment of the cells: Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. Each concentration of the test item, medium control, positive and DMSO control was tested in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).
Staining of the cells: The triplicates of each test item-treated and not test item treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). All solutions were kept light protected at 2-8 °C or on ice during the staining and analysis procedure. The cells were gently mixed by hand and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).
Sample preparation for measurement: After staining with the antibodies, the cells were washed twice (2-8 °C) with 2 mL FACS buffer 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-aminoactinomycin D (7-AAD) solution were added.
Flow cytometry acquisition: The expression of cell surface antigens (CD54, CD86) was analyzed by flow cytometry. 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.
Acquisition: A total of 10,000 living cells were analyzed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50 % (due to diffuse labelling of cytoplasmic structures that are generated due to cell membrane destruction).
Data analysis and interpretation:
The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical:
RFI (%) = 100 x (MFI of test item treated cells - MFI of test item treated isotope control cells) / (MFI of solvent control cells - MFI of solvent isotope control cells), where MFI is geometric mean fluorescent intensity
The cell viability is calculated as follows:
Cell viability (%) = 100 x (Mean cytotoxicity of solvent control cells) / (Mean cytotoxicity of the test item treated cells), where Mean cytotoxicity is the mean of geometric mean (7-AAD) isotype control, geometric mean (7-AAD) CD54 and geometric mean (7-AAD) CD86.
Acceptability criteria of the h-CLAT assay:
The study is considered as valid, if the following criteria are met:
• Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the 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 medium and DMSO controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• For the test item resulting in negative outcome, the cell viability at the 1.2 × CV75 should be less than 90%. (If the cell viability at the 1.2 × CV75 is more than 90% for a positive tested test item, the data will be acceptable. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test item are accepted independent by the cell viability.)
• The cell viability of at least 4 doses in each experiment should be ≥50%.

Evaluation of results:
The test item is tested in two independent runs. If the RFI of CD86 is ≥ 150% or if the RFI of CD54 is ≥ 200% in both independent run data, the test item is considered to be a sensitiser. Otherwise, it is considered to be a non-sensitiser. In case of different results in both runs, a third run has to be performed. If the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent run data, or if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data, the test item is considered to be a sensitiser. Otherwise it is considered to be a non-sensitiser.

Positive control results:

The RFI value for CD54 of the positive control (3 µg/mL DNCB) in the first run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the RFI values for CD54 and CD86 of the positive control (2 µg/mL DNCB) exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. The RFI values of the positive controls (DNCB) for CD54 and CD86 of the second run exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

Key result

Run / experiment:

other: 1, CD86

Parameter:

other: % RFI

Value:

171.1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

(>150.0%) at the concentration of 2170 ug/mL

Key result

Run / experiment:

other: 1, CD54

Parameter:

other: % RFI

Value:

171.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

(<200.0%)

Key result

Run / experiment:

other: 2, CD86

Parameter:

other: % RFI

Value:

159.8

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

(>150.0%) at the concentration of 1046 ug/mL

Key result

Run / experiment:

other: 2, CD54

Parameter:

other: % RFI

Value:

205.6

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

(>200.0%) at the concentration of 727 ug/mL

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- Acceptance criteria met for positive control: The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%. The RFI value for CD54 of the positive control (3 µg/mL DNCB) in the first run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the RFI values for CD54 and CD86 of the positive control (2 µg/mL DNCB) exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. The RFI values of the positive controls (DNCB) for CD54 and CD86 of the second run exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

Results of the XTT assay:

1st test:

		Microscopic Evaluation
Test Group	Concen-tration [µg/mL]	Cytotoxicity	Mean Ab-sorbance*	Standard-Deviation	Chem. Blank	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.586	0.038	0.214	0.372	109.38
Solvent Control	-	no	0.552	0.018	0.212	0.340	100.00
Test Item	39.1	no	0.613	0.026	0.213	0.400	117.76
	78.1	no	0.606	0.032	0.210	0.396	116.62
	156.3	no	0.604	0.037	0.216	0.388	114.04
	312.5	no	0.561	0.026	0.214	0.347	102.23
	625	no	0.566	0.020	0.215	0.351	103.32
	1250	no	0.572	0.041	0.213	0.359	105.52
	2500	no	0.489	0.086	0.215	0.274	80.72
	5000	yes	0.312	0.034	0.216	0.096	28.19

Shaded test groups:            cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

*             mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium controlwas 91.42%.

The CV75 value of the first XTTtest: 2772.2 µg/mL

2nd test:

		Microscopic Evaluation
Test Group	Concen-tration [µg/mL]	Cytotoxicity	Mean Ab-sorbance*	Standard-Deviation	Chem. Blank	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.590	0.029	0.211	0.379	93.66
Solvent Control	-	no	0.610	0.057	0.205	0.405	100.00
Test Item	39.1	no	0.686	0.103	0.213	0.473	116.81
	78.1	no	0.653	0.055	0.212	0.442	109.07
	156.3	no	0.661	0.056	0.209	0.452	111.53
	312.5	no	0.645	0.046	0.211	0.434	107.14
	625	no	0.582	0.057	0.210	0.372	91.81
	1250	no	0.581	0.045	0.212	0.369	91.07
	2500	no	0.324	0.100	0.211	0.113	27.99
	5000	yes	0.233	0.013	0.213	0.020	4.86

Shaded test groups:            cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

*             mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium controlwas 106.77%.

The CV75 value of the second XTTtest: 1568.4 µg/mL

The mean CV75 value of both XTTtests: 2170.3 µg/mL

Results of the h-CLAT test:

1st test run:

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	250.3*	374.8*	57.4
	3.0	195.4^#	307.4*	57.7
Test Item	727	169.3	139.8	80.2
	872	169.3	134.3	84.5
	1046	127.9	89.1	102.1
	1256	145.7	120.9	87.5
	1507	149.3	140.8	87.2
	1808	170.0	136.8	86.3
	2170	171.4	171.1*	76.2
	2604	355.7*	2932.8*	6.3

Shaded test groups:            cell viability below 50%, are excluded from the evaluation

^#CD54 RFI value of the positive control (3.0 µg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200%).

*   RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥150% and CD54≥200%).

2nd test run:

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	258.1*	374.6*	71.1
	3.0	423.1*	720.6*	67.8
Test Item	727	205.6*	102.5	96.8
	872	272.2*	115.6	100.7
	1046	350.9*	159.8*	91.6
	1256	592.6*	379.5*	76.2
	1507	788.9*	936.1*	44.6
	1808	1041.7*	1789.3*	27.2
	2170	1082.4*	2592.6*	23.3
	2604	992.6*	5097.5*	12.2

Shaded test groups:            cell viability below 50%, are excluded from the evaluation

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86≥150% and CD54≥200%).

Interpretation of results:

other: h-CLAT was positive

Conclusions:

In a GLP-compliant guideline study, the test substance was found to be positive in the in vitro h-CLAT test, indicating a possible skin sensitizing potential of the substance.

Executive summary:

The GLP-compliant in vitro Human Cell Line Activation Test (h-CLAT) was performed in accordance with OECD guideline 442E to assess one part of the skin sensitisation mechanism. The substance is dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The log Kow of the test substance is 3.6 while the applicability domain of the h-Clat for physico-chemical properties is 3.5. The substance being a liquid with a low molecular weight this will not have affected the results. The validity criteria were met, e.g. the values obtained for controls.The following concentrations of the test item (solved in culture medium) were tested in the main experiment (h-CLAT) based on the results of two XTT tests: 727, 872, 1046, 1256, 1507, 1808, 2170 and 2604 µg/mL. The test substance was tested in two valid independent runs. The RFI of CD86 and CD54 were ≥ 150% and ≥ 200%, respectively in at least one concentration of the second run and the RFI of CD86 was ≥ 150% in the second highest concentration of the first run. Therefore, the substance is considered positive in this h-Clat assay.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 - 12 May 2017

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)

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

Controls:
Positive control: ethylene dimethacrylate glycol, 7.81-250 µM, tested in triplicate
Negative control: DMSO (vehicle) (1% in exposure medium), 18 wells/plate
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values

Number of replicates: two independent experiments, each concentration tested in triplicate for the luciferase activity measurements, one parallel replicate for MTT cell viability assay.

Test system:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.
Cells were used at passage 21 for experiment 1 and at passage 23 for experiment 2. For testing cells were 80-90% confluent.
One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator.

Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 59 – 86%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 – 37.5°C).

Test item preparation:
No correction was made for the composition/purity of the test item.
The test item was dissolved in DMSO to a final concentration of 200 mM. This concentration was selected as highest concentration for the main assay (highest dose required in the guideline).
The stock and spike solution were diluted 25-fold with exposure medium (final concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.977 µM (final concentration DMSO of 1%).
All concentrations of the test item were tested in triplicate. No precipitate was observed at the start and end of the treatment in the MTT assay plates.

Media:
Basic medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.
Maintenance medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 µg/ml).
Exposure medium
Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

Treatment of cells:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at .37±1.0°C in the presence of 5% CO2.

Luciferase acitivity measurement:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time one second).

Cytotoxicity assessment:
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed (e.g. by adding 10% SDS solution to each well) overnight. After shaking, the absorption is measured with the TECAN Infinite® M200 Pro Plate Reader.

Data analysis:
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical 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) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.

Equation 1: Fold induction= (Lsample - Lblank)/(Lsolvent - Lblank)
Where:
Lsample is the luminescence reading in the test chemical well
Lblank is the luminescence reading in the blank well containing no cells and no treatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative) control

The EC1.5 is calculated by linear interpolation according to Equation 2, and the overall EC1.5 is calculated as the mean of the individual repetitions.

Equation 2: EC1.5 = (Cb - Ca) x [(1.5 - Ia) / (Ib - Ia)] + Ca
Where:
Ca is the lowest concentration in μM with > 1.5 fold induction
Cb is the highest concentration in μM with < 1.5 fold induction
Ia is the fold induction measured at the lowest concentration with > 1.5 fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with < 1.5 fold induction (mean of three replicate wells)

Viability is calculated by Equation 3:
Equation 3: Viability = 100 x (Vsample - Vblank) / (Vsolvent - V blank)
Where:
Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent (negative) control
Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.

In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using 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.

Data intepretation:
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 µM).
• The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
• Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 6 wells tested. If the variability is higher, results should be discarded.

Negative results obtained with concentrations < 1000 µM should be considered as inconclusive.

Key result

Run / experiment:

other: Experiment 1, 1000 µM

Parameter:

other: Imax

Value:

1.16

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: first criterion of positive result is not met

Key result

Run / experiment:

other: Experiment 2, 62.5 µM

Parameter:

other: Imax

Value:

1.15

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: first criterion for positive result is not met

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (16.8% and and 15.4% in experiment 1 and 2, respectively).

- Acceptance criteria met for positive control: The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. The EC1.5 of the positive control was between 5 and 125 µM (24.1 µM and 119.2 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (20.9-fold and 2.19-fold in experiment 1 and 2, respectively).

Test substance showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.16-fold and 1.15-fold in experiment 1 and 2 respectively. Diola BHT is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations of ≥1000 µM with a cell viability of >70% compared to the vehicle control.

Summary tables

Table1          Overview of the luminescence induction and cell viability of the test substance in Experiment 1 and 2

Concentration (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.50	125	250	500	1000	2000
Exp 1 luminescence	0.84	0.54	0.95	0.85	0.90	0.92	0.92	0.93	0.93	1.10	1.16	1.14
Exp 1 viability (%)	102.0	111.7	104.2	105.7	98.5	103.6	104.0	107.7	115.3	119.4	121.3	123.8
Exp 2 luminescence	0.91	0.79	0.84	0.83	0.85	0.88	1.15	0.96	0.91	0.85	0.92	0.92
Exp 2 viability (%)	114.5	110.7	115.6	108.1	113.3	104.8	105.1	112.4	124.3	121.4	126.4	113.7

Table 2.

Overview luminescence induction and cell viability for the positive control Ethylene dimethacrylate glycol in Experiments 1 and 2

Concentration (µM)	7.81	15.6	31.3	62.5	125	250
Exp 1 luminescence	1.07	1.40	1.58	2.45	4.36	20.88^***
Exp 1 viability (%)	112.3	133.3	131.6	140.7	150.2	133.7
Exp 2 luminescence	0.92	1.00	1.20	1.18	1.53^***	2.19^***
Exp 2 viability (%)	120.4	122.9	134.8	147.9	132.2	100.1

*** p<0.001 Students t-test

Table3. Overview of EC_1.5, I_max, IC₃₀and IC₅₀values

	EC1.5 (µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	NA	1.16	NA	NA
Test item Experiment 2	NA	1.15	NA	NA
Pos Control Experiment 1	24.1	20.88	NA	NA
Pos Control Experiment 2	119.2	2.19	NA	NA

N.A. = Not applicable

Table 4. Historical control data

	Positive control
	EC1.5	Imax
Range	5.7 – 109.0	1.79 – 11.13
Mean	43.2	3.51
SD	29.0	1.60
n	46	46

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2016 to April 2017.

 

Interpretation of results:

other: KeratinoSens is negative

Conclusions:

In a GLP-compliant guideline study, the test substance did not cause a biologically relevant induction in luciferase activity in Keratinosens assay. Based on this, the test substance is considered to give a negative result under the experimental conditions of this assay.

Executive summary:

The GLP-compliant in vitro KeratinoSens (ARE-Nrf2 luciferase reporter) assay was performed in accordance with OECD guideline 442D to assess anti-oxidant/electrophilic activity of the substance being one part of the skin sensitisation mechanism. The test substance falls within the applicability domain of the assay. Two independent experiments were performed. The test substance was tested in a concentration range of test concentrations of 0.98 – 2000 μM (2-fold dilution series). The acceptance criteria were met. Test substance showed no toxicity (no IC30 and IC50 value). Also no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.16-fold and 1.15-fold in experiment 1 and 2, respectively. Test substance is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed at test concentrations of ≥1000 µM with a cell viability of >70% compared to the vehicle control. Overall the test substance is considered as negative in the KeratinoSens assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitization of the test substance was tested in a battery of one in chemico and two in vitro assays (DPRA, KeratinoSens and h-CLAT, respectively).

DPRA:

In a GLP-compliant OECD guideline 442C study, Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity of the substance by protein depletion being one part of its sensitisation mechanism. In the cysteine and lysine reactivity assay all analytical acceptance criteria of the test were met. The test substance caused -1.31% (SD 0.81%) and -0.821% (SD 0.74%) cysteine and lysine peptide depletion, respectively. These results are categorised as “no to minimal reactivity” based on the DPRA prediction model and is thus considered to be negative in the DPRA.

KeratinoSens:

The GLP-compliant in vitro KeratinoSens (ARE-Nrf2 luciferase reporter) assay was performed in accordance with OECD guideline 442D to assess anti-oxidant/electrophilic activity of the substance being one part of the skin sensitisation mechanism. The test substance falls within the applicability domain of the assay. Two independent experiments were performed. The test substance was tested in a concentration range of test concentrations of 0.98 – 2000 μM (2-fold dilution series). The acceptance criteria were met. Test substance showed no toxicity (no IC30 and IC50 value). Also no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.16-fold and 1.15-fold in experiment 1 and 2, respectively. Test substance is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed at test concentrations of ≥1000 µM with a cell viability of >70% compared to the vehicle control. Overall the test substance is considered as negative in the KeratinoSens assay.

h-CLAT:

The GLP-compliant in vitro Human Cell Line Activation Test (h-CLAT) was performed in accordance with OECD guideline 442E to assess one part of the skin sensitisation mechanism. The substance is dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The log Kow of the test substance is 3.6 while the applicability domain of the h-CLAT for physico-chemical properties is 3.5. Because the substance is a liquid with a low molecular weight, this will not affect the results. The validity criteria were met, e.g. the values obtained for controls. The following concentrations of the test item (solved in culture medium) were tested in the main h-CLAT experiment (based on the results of two XTT tests): 727, 872, 1046, 1256, 1507, 1808, 2170 and 2604 µg/mL. The test substance was tested in two valid independent runs. The RFI of CD86 and CD54 were ≥ 150% and ≥ 200%, respectively in at least one concentration of the second run and the RFI of CD86 was ≥ 150% in the second highest concentration of the first run. Therefore, the substance is considered positive in this h-Clat assay.

Overall conclusion:

Overall, one in chemico and two in vitro assays were conducted. The DPRA (in chemico) and the KeratinoSens (in vitro) were negative. The h-CLAT in vitro assay was positive. Based on the negative results in 2 out of 3 tests, the overall conclusion of these tests is that the substance has no skin sensitizing potential in accordance with Bauch et al., 2012.

Reference: Bauch, C., Kolle, S.N., Ramirez, T., Eltze, T., Fabian E., Mehling, A., Teubner, W., van Ravenzwaay, B., Landsiedel, R., 2012, Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials, Regul. Toxicol. Pharmacol., 63, 489–504.

Justification for classification or non-classification

The test substance produced negative results in the DPRA and KeratinoSens (ARE-Nrf2 luciferase reporter assay). Based on the testing strategy presented, classification of this substance for skin sensitisation is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its updates.