Octahydro-2H-1-benzopyran-2-one

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): not sensitizing

Skin sensitization (maximization test, OECD 406): not 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:

14 November 2016 -19 November 2016

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:

d.d. 15/2/2016

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.

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.

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)]

Acceptance criteria for analytical measurements are presented in Table 1 in the section "Any other information on materials and methods incl. tables". Information on the interpretation of the results is presented in the same section in Table 2.

Positive control results:

72.8% depletion (CV 1.29%, n = 3) and 59.1% depletion (CV 0.85%, 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:

-1.85

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 negative control: not applicable. Reference (stability) controls and precision controls of both peptides were met (CV 1.85%, n = 6 and CV 0.31%, n = 6, for cysteine and lysine, respectively, at 0.50 mM).
- Acceptance criteria met for positive control: yes, 72.8% depletion (CV 1.29%, n = 3) and 59.1% depletion (CV 0.85%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.
- Acceptance criteria met for variability between replicate measurements: yes, CV 0.03% and 1.58%, respectively, for cysteine and lysine depletion by the test item.

TEST SUBSTANCE RESULTS:

Mean depletion of -1.85% and -13.2% was observed for the test substance with cysteine and lysine peptides, respectively. As co-elution was observed with the lysine peptide, only the result for cysteine peptide is reported using the depletion model (see section "Any other information on materials and methods incl. tables"). With the test substance not reacting with the cysteine 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 1:10 / Lysine 1:50 prediction model.

Executive summary:

In a GLP-compliant OECD guideline 442C study, Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity and sensitizing potential of the test substance. The test substance was within the applicability domain of the assay.

Solutions of the test substance were successfully analyzed by the validated DPRA analytical method. In the lysine reactivity assay, there was co-elution of the test item with the lysine peak, therefore no conclusions on reactivity could be drawn from this assay according to the test guideline.

In the cysteine reactivity assay all analytical acceptance criteria of the test were met. Therefore the conclusions were based only on the cysteine results.

The test substance caused -1.85% cysteine peptide depletion and was classified as “no to minimal reactivity” based on the DPRA prediction model and was 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:

16 November, 2016 - 25 November, 2016

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 9 in both XTT assays and 10 and 11 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): 273.5, 328.2, 393.8, 472.6, 567.1, 680.6, 816.7 and 980 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 ± 1 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 2 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 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%.

Key result

Run / experiment:

other: 1

Parameter:

other: % RFI; CD54

Value:

225

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

At 680.6 umol the value is > 200

Key result

Run / experiment:

other: 1

Parameter:

other: % RFI; CD86

Value:

153

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

AT 680.6 umol the value is > 150.

Key result

Run / experiment:

other: 2

Parameter:

other: % RFI; CD54

Value:

411

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

At 980 umol the value is > 200.

Key result

Run / experiment:

other: 2

Parameter:

other: % RFI; CD86

Value:

173

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

At 980 umol the value is > 150.

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%.

Results of the XTT assay:

1st test:

			Microscopic		Photometric
			Evaluation		Evaluation
Test Group	Concentration		Cytotoxicity	Mean Ab-	Standard-	Chem.		Absorbance in % of
	[µg/mL]			sorbance*	Deviation	Blanks		Solvent Control**
Medium Control	-		no	0.722	0.043	0.252		85.81
Solvent Control	-		no	0.804	0.097	0.257		100.00
	39.1		no	0.815	0.081	0.255		102.43
	78.1		no	0.889	0.104	0.262		114.55
	156.3		no	0.990	0.168	0.265		132.40
	312.5		no	0.881	0.105	0.255		114.56
Test Item
	625		no	0.760	0.080	0.254		92.42
	1250		no	0.629	0.069	0.246		70.05
	2500		yes	0.261	0.010	0.237		4.33
	5000		yes	0.235	0.005	0.220		2.69
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 control was 116.6%.

 

The CV75 value of the first XTT test: 1111.7 µg/mL

2nd test:

			Microscopic				Photometric
			Evaluation				Evaluation
Test Group	Concentration		Cytotoxicity		Mean Ab-		Standard-		Chem.		Absorbance in % of
	[µg/mL]				sorbance*		Deviation		Blanks		Solvent Control**
Medium Control	-		no	0.580		0.017		0.232			97.86
Solvent Control	-		no	0.593		0.021		0.237			100.00
	39.1		no	0.586		0.033		0.227			100.75
	78.1		no	0.584		0.025		0.231			99.09
	156.3		no	0.592		0.029		0.238			99.49
	312.5		no	0.545		0.037		0.232			88.09
Test Item
			no		0.474		0.032		0.230		68.47
	625
	1250		no		0.370		0.036		0.219		42.51
	2500		yes		0.199		0.018		0.214		-4.34
	5000		yes		0.187		0.012		0.201		-3.91
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 control was 102.3%.

 

The CV75 value of the second XTT test: 521.0 µg/mL

 

The mean CV75 value of both XTT tests: 816.4 µg/mL

Results of the h-CLAT test:

1st test run:

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability
				(%)
Medium Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
DMSO Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
Positive Control (DNCB)	2.0	CD 54	494.7*	72.5
		CD 86	327.2*
	3.0	CD 54	507.7*	79.1
		CD 86	393.7*
Test Item	273.5	CD 54	149.6	98.6
		CD 86	126.4
	328.2	CD 54	130.7	98.8
		CD 86	122.4
	393.8	CD 54	143.3	98.6
		CD 86	132.8
	472.6	CD 54	163.0	97.7
		CD 86	134.4
	567.1	CD 54	274.0*	97.6
		CD 86	148.0
	680.6	CD 54	225.2*	96.7
		CD 86	152.8*
	816.7	CD 54	278.7*	96.8
		CD 86	184.8*
	980	CD 54	329.9*	93.1
		CD 86	212.8*

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

2nd test run:

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability (%)
Medium Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
DMSO Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
Positive Control (DNCB)	2.0	CD 54	225.9*	66.7
		CD 86	536.8*
	3.0	CD 54	326.7*	64.9
		CD 86	451.5*
Test Item	273.5	CD 54	136.0	94.6
		CD 86	111.9
	328.2	CD 54	140.7	92.4
		CD 86	100.0
	393.8	CD 54	153.5	93.5
		CD 86	115.7
	472.6	CD 54	158.1	93.4
		CD 86	117.6
	567.1	CD 54	148.8	87.7
		CD 86	95.0
	680.6	CD 54	208.1*	88.9
		CD 86	93.1
	816.7	CD 54	275.6*	84.7
		CD 86	103.8
	980	CD 54	410.5*	88.0
		CD 86	173.0*

*  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 the skin sensitising potential of the test substance dissolved in culture medium when administered to THP-1 cells for 24 ± 1 hours. The test substance falls within the applicability domain of the assay. 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 HTT tests:  273.5, 328.2, 393.8, 472.6, 567.1, 680.6, 816.7 and 980 µg/mL. The test substance was tested in 2 valid independent runs. The RFI of CD86 and CD54 was greater than 150% and 200%, respectively, in at least one dose of both independent run data. Therefore, the test item is considered to have an indication of a skin sensitizing potential.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 November 2016 - 02 December 2016

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 19 for experiment 1 and at passage 21 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 60 – 100%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 –38.4°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. The compound formed a clear colourless solution at 200 mM. From the stock 11 spike solutions in DMSO were prepared (2-fold dilution series): 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, 0.391, 0.195 and 0.0977 mM.
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. All formulations formed a clear solution. 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, 2000 µM

Parameter:

other: Imax

Value:

1.66

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: first criterion of positive result is met

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5, µM

Value:

1 724

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: third criterion of positive result is not met

Key result

Run / experiment:

other: Experiment 2, 2000 µM

Parameter:

other: Imax

Value:

0.87

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% (9.0% and 10.4% in experiment 1 and 2, respectively).
- Acceptance criteria met for positive control: yes. 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 (109 µM and 106 µM in experiment 1 and 2, respectively). In the first experiment, the induction at 250 µM was with 1.79 just below 2-fold, nevertheless a clear dose response was observed. In experiment 2 the induction at 250 µM was higher than 2-fold and a dose response was observed.

- Acceptance criteria met for variability between replicate measurements: yes. The test substance showed slight toxicity in experiment 1 (an IC30 of 1928 µM and no IC50) and no toxicity in experiment 2 (no IC30 and IC50 value). Although an induction of the luminescence activity >1.5-fold (Imax = 1.66-fold) was observed in experiment 1, the EC1.5 was calculated to be 1724 µM which is ≥ 1000 µM and therefore the test substance is classified as negative by the KeratinoSens prediction model in experiment 1. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in experiment 2 and the Imax was 0.87-fold. Overall the test substance is classified as negative in the KeratinoSens(TM) assay

Summary tables

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

Concentration (µM)	0.977	1.95	3.91	7.81	15.63	31.25	62.50	125	250	500	1000	2000
Exp 1 luminescence	0.82	0.82	0.96	0.84	0.92	0.98	0.89	0.86	1.02	1.00	1.09	1.66***
Exp 1 viability (%)	90.3	99.4	108.9	111.7	113.4	115.7	101.1	117.0	83.4	76.1	80.8	69.2
Exp 2 luminescence	0.70	0.57	0.60	0.64	0.65	0.63	0.66	0.62	0.65	0.76	0.74	0.87
Exp 2 viability (%)	120.3	155.8	161.3	143.7	133.0	142.7	151.9	156.1	163.1	160.5	171.4	115.6

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	0.90	1.00	1.25	1.26	1.58***	1.79***
Exp 1 viability (%)	95.6	94.2	96.2	103.4	97.9	97.6
Exp 2 luminescence	1.49	1.23	1.37	1.27	1.60***	2.48***
Exp 2 viability (%)	113.2	111.4	120.6	110.3	141.9	125.7

*** p<0.001 Students t-test

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

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Bicyclononalactone Experiment 1	1724	1.66	1928	NA
Bicyclononalactone Experiment 2	NA	0.87	NA	NA
Pos Control Experiment 1	109	1.79	NA	NA
Pos Control Experiment 2	106	2.48	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 in 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 the skin sensitising potential of the test substance. 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 0.977 – 2000 μM (2-fold dilution series). The acceptance criteria were met. The test substance showed slight toxicity in experiment 1 (an IC₃₀of 1928 µM and no IC₅₀) and no toxicity in experiment 2 (no IC₃₀and IC₅₀value). Although an induction of the luminescence activity >1.5-fold (I_max= 1.66-fold) was observed in experiment 1, the EC_1.5 was calculated to be 1724 µM which is ≥ 1000 µM and therefore the test substance is classified as negative by the KeratinoSens^TMprediction model in experiment 1. No biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations in experiment 2 and the I_maxwas 0.87-fold. Overall the test substance is classified as negative in the KeratinoSens^TMassay.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29 January, 1980 - 08 May, 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

(1981)

Deviations:

yes

Remarks:

no positive control animals to demonstrate the sensitivity and reliability of the test

GLP compliance:

no

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An LLNA skin sensitisation study does not need to be conducted because the skin sensitization of the test substance was tested in a battery of two in vitro, one in chemico test (KeratinoSens, h-CLAT and DPRA assays) and one in vivo study (guinea pig maximization assay).

Specific details on test material used for the study:

- Description of test substance: Octahydrocoumarin
- Appearance: clear, colourless liquid

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Environmental Safety Division Unilever
- Weight at Start: males: 352-382g; females: 356-410g

ENVIRONMENTAL CONDITIONS (check details)
No data.

Route:

intradermal

Vehicle:

other: 1% acetone/0.01% Dobs/saline

Concentration / amount:

1%

Day(s)/duration:

Single

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol

Concentration / amount:

50%

Day(s)/duration:

The patch is held in place for 48 hours. Fourteen days thereafter the animals were challenged.

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol

Concentration / amount:

10%

Day(s)/duration:

24 and 48 hours

Adequacy of challenge:

highest non-irritant concentration

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol

Concentration / amount:

10%

Day(s)/duration:

24 and 48 hours

Adequacy of challenge:

highest non-irritant concentration

No.:

#3

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol

Concentration / amount:

10%

Day(s)/duration:

24 and 48 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Test animals: 10 (6 males, 4 females)
Control animals: 8 (4 males, 4 females)

Details on study design:

RANGE FINDING TESTS
- Intradermal injections:
Intradermal injections (0.1 mL/site) were made into the clipped flank of four males at concentrations of 2.5 and 1% of the test substance in 0.01% Dobs/saline. Twenty-four hours later the reactions were examined for size in millimetres (lenght and breadth), erythema and oedema.
As pale pink oedema was observed with 1% in the four animals, this concentration was selected as the intradermal induction concentration.

- Epidermal application:
Eight millimetre diameter filter paper patches in 11mm aluminium patch test cups were saturated with concentrations of 50, 25 and 10% in ethanol and the cups were applied to the shaved flanks of four males and weighing approximately 450g. The patches were held in place by adhesive plaster wound around the trunk. The patches were removed 24 hours after application and the treatment sites examined 24 and 48 hours after removal of the patches.
The resulting reactions were scored for irritation on a scale from 0 to +++. For shoulder induction, a concentration giving slight but definite irritation was selected. The highest concentration which causes no visible irritation was selected for challenge.
At the topical induction of 50% test substance, one animal showed barely perceptible erythema 24 hours after treatment 48 hours after treatment no effects were observed. The other 3 animals showed no effects 24 and 48 hours after treatment. For the challenge 10% was determined to be the highest non-irritant concentration.

MAIN STUDY
A. INDUCTION EXPOSURE
1) Intradermal injections
- Concentration: 1%
- Site: the dorsal scapular region
Three pairs of intradermal injections (0.1 mL/site):
1) Freund's complete adjuvant (FCA) 50:50 with 1% acetone/0.01% Dobs/saline
2) Test substance at 1% in 1% acetone/0.01% Dobs/saline
3) Test substance at 2% in 1% acetone/0.01% Dobs/saline in a 50:50 mixture of FCA with 1% acetone/0.01% Dobs/saline

2) Topical applications one week after the injections:
- Concentration: 50% (in ethanol)
- Amount: saturated patch (control animals: ethanol only)
- Area: 8 cm2
- Exposure period: 48 hours (occlusive)
- Readings: 24 and 48 hours after patch removal

Controls:
Treated controls: At the same time as the test animals were selected, 4 males were selected as treated controls for the first challenge. They were given a mock induction treatment at the same time and in the same way as for the test animals except that test substance was omitted from the injection and application preparations. At first challenge they were treated in exactly the same way as the test substance.
Untretaed controls: At every challenge in the test 4 previously untreated animals of the same sex and weighing approximately the same as the test animals at that challenge were treated in exactly the same way as the test animals. When animals of similar weight to the test animals were not available, those as close as possible in weight were selected from stock.

B. CHALLENGE EXPOSURE (control and test group)
- Day of challenge: Fourteen days after application of the induction patch. One week after the first challenge a second challenge was made on the opposite flank exactly as for the first challenge. Further challenges and cross challenges may be made on alternate flanks at weekly or greater intervals as required.
- Concentrations: 10%
- Exposure period: 24 hours (occlusive)
- Sites: clipped and shaved flank
- Amount: saturated patch
- Readings: 24 and 48 hours after patch removal

A reaction was considered to be a sensitization reponse if: response produced scattered, mild erythema (faint pink) or greater and there were no reactions in controls or no reaction greater than barely perceptible erythema in controls.

Positive control substance(s):

no

Key result

Reading:

other: 1st reading and 3rd reading

Hours after challenge:

24

Group:

negative control

Dose level:

10%

No. with + reactions:

0

Total no. in group:

4

Key result

Reading:

other: 1st reading and 3rd reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

other: 1st reading and 3rd reading

Hours after challenge:

48

Group:

negative control

Dose level:

10%

No. with + reactions:

0

Total no. in group:

4

Key result

Reading:

other: 1st reading and 3rd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

2nd reading

Hours after challenge:

24

Group:

negative control

Dose level:

10%

No. with + reactions:

0

Total no. in group:

4

Key result

Reading:

2nd reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

1

Total no. in group:

10

Remarks on result:

other: at 24 hours 1 animal showed scattered, mild erythema (faint pink).

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

10%

No. with + reactions:

0

Total no. in group:

4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

1

Total no. in group:

10

Remarks on result:

other: at 48 hours 1 other animal showed spots of erythema.

Interpretation of results:

other: Not sensitising

Remarks:

According to Regulation (EC) No. 1272/2008 and its updates.

Conclusions:

In a guinea pig maximisation test performed equivalent to OECD 406 guideline, the substance is not considered a skin sensitiser.

Executive summary:

The skin sentisation potential of the substance was investigated by performing a guinea pig maximisation test equivalent to OECD 406 guideline. A concentration of 1% was used for the intradermal induction, 50% for the epidermal induction and 10% for the challenge. No biologically relevant effects were observed in each of the 3 challenges (only barely perceptible erythema was observed in some animals, which was also observed in some control animals).The substance was not considered to be a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitization potential is assessed with in silico, in vitro and in vivo information and is shortly summarized. Thereafter the executive summaries of the individual studies are presented.

In silico: The substance does not have a protein binding potential according to the OECD Toolbox.

In vitro: The substance tested was negative in the DPRA, negative in the Keratinosens but positive in The h-CLAT. In absence of protein binding (DPRA) and specific signals for skin sensitisation (Keratinosens), the potential of forming antibodies (h-CLAT) in isolation, will not present a skin sensitization potential.

In vivo: A maximization test was performed which was negative but with some limitations.

 

Overall conclusion: Bicyclononalactone does not have a skin sensitization potential based on the key information of three in vitro skin sensitization tests. The ITS proposed by Bauch et al. 2012) presents that when 2/3 in vitro results are negative the substance can be considered a non-skin sensitiser. In addition, there is no structural alert for skin sensitization potential and the negative in the Maximisation test.

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.

 

DPRA: In the DPRA assay, conducted according to OECD TG 442C, the test substance caused -1.85% cysteine peptide depletion and was classified as “no to minimal reactivity” based on the DPRA prediction model. As a co-elution of the test substance and lysine peptide was observed, only the results with cysteine peptide were used for the prediction model. Based on the observed result the test substance was considered to be negative in the DPRA.

Keratinosens: In the KeratinoSens Assay (ARE-Nrf2 luciferase reporter assay), conducted according to OECD TG 442D, two independent experiments were conducted. The maximal induction of luciferase activity (Imax) was 1.66 in Experiment 1. As this Imax was only reached at a test substance concentration of1724 µM, which is > 1000 µM, the test substance was classified as negative by the KeratinoSens prediction model in experiment 1. No biologically relevant induction of the luciferase activity was measured at any of the test concentrations in experiment 2 and the Imax was 0.87-fold. Overall the test substance was classified as negative in the KeratinoSens assay under the experimental conditions described in the report.

h-CLAT: The GLP-compliantin vitroHuman Cell Line Activation Test (h-CLAT) was performed in accordance with OECD guideline 442E to assess the skin sensitising potential of the test substance dissolved in culture medium when administered to THP-1 cells for 24 ± 1 hours. The test substance falls within the applicability domain of the assay. 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 HTT tests: 273.5, 328.2, 393.8, 472.6, 567.1, 680.6, 816.7 and 980 µg/mL.The test substance was tested in 2 valid independent runs.The RFI of CD86 and CD54 was greater than 150% and 200%, respectively,in at least one dose of both independent run data. Therefore, the test item is considered tohave an indication of a skin sensitizing potential.

Maximisation test: In an in vivo maximization test, conducted according to OECD TG 406 but limitedly reported on test substance identity and presence of a positive control. The intradermal induction with 1% solution of the test substance in acetone/0.01% Dobs/saline and the epidermal induction with 50% test substance in ethanol was carried out. The intradermal concentration was clearly irritant in 4/4 animals. The epidermal induction was slightly irritant (1/4 animals). The maximum non-irritant concentration was 10% and used for the challenge. No biologically relevant effects were observed following three challenges with 10% test substance in ethanol(only barely perceptible erythema was observed in some animals, which was also observed in some control animals). The study had some limitations; in particular, a positive control was not included and no information on the test substance purity was reported. Nevertheless, the substance was not considered to be a skin sensitiser under the conditions reported in the study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Respiratory sensitisation can be assessed using human data such as indicated in R7.3.5.2 of the ECHA guidance (2015) that indicate respiratory reactions e. g. from consumer experience or occupational exposure. In case no such data are available the respiratory sensitisation can be assessed using the integrated evaluation strategy for respiratory sensitisation data in the ECHA guidance (R7A, Fig. 7.3-2, 2015), which says that if the substance is not a skin sensitiser, it is unlikely to be a respiratory sensitiser.

Justification for classification or non-classification

Based on the presented information the substance is not a skin sensitiser and not a respiratory sensitiser. Therefore classification of this substance for skin and respiratory sensitisation is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its updates.