trans-1-[4-(4-Propylcyclohexyl)-1-cyclohexen-1-yl]-4- (trifluoromethyl)-benzene

Endpoint summary

Description of key information

Skin sensitisation was investigated using in vitro studies and based on the inconclusive results obtained a further in vivo OECD 429 assay. The following results have been obtained:

• OECD 442C: negative but inconclusive due to precipitation
• OECD 442D: technically impossible due to limited solubility
• OECD 442E: negative, but cannot be used due to logP exceeding threshold of 3.5

The in vitro data are insufficient to conclude on the endpoint of skin sensitisation. Therefore, a further in vivo study was performed giving a clear negative response.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Feb 13, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Remarks:

Preliminary study to determine the solubility of the test item

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde für Gesundheit und Verbraucherschutz, Freie Handelsstadt Hamburg

Type of study:

activation of keratinocytes

Details on the study design:

According to OECD 442D, the KeratinoSens test method is applicable to the testing of chemicals that are soluble or form a stable dispersion, either in water or DMSO.
The preliminary test (testing of the test material for solubility or suitability in the assay) was thus carried out with water and DMSO.

Key result

Run / experiment:

other: 1

Parameter:

other: solubility screening

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: insoluble in test solutions

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item appeared to be insoluble in the required concentration in water. The test item was insoluble in DMSO at the required stock solution concentration of 200 mM and formed no stable suspension after addition of treatment medium. Since the KeratinoSens assay is not suitable for test items which precipitate into non stable suspensions or emulsions (defined by OECD 442D) the assay has to be considered as being technically impossible.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Feb 14, 2020 - Sep 14, 2020

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)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared in a solution of 20% acetonitrile:buffer using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide. The following calibration solutions were prepared from the peptide stock solution of 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 blank of the dilution buffer was also included in the standard calibration curve for both peptides. The blank is 25% acteonitrile:buffer solution with phosphate buffer pH 7.5 for the cysteine peptide and with ammonium acetate buffer pH 10.2 for the lysine peptide without peptide.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and test item stock solutions were diluted with the cysteine peptide stock solution so as to prepare solutions containing 500 µM cysteine and 5 mM of Cinnamaldehyde or 5 mM of the test item. For the co-elution control, buffer solution was used in place of the cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and test item stock solutions were diluted with the lysine peptide stock solution so as to prepare solutions containing 500 µM lysine and 25 mM of Cinnamaldehyde or 25 mM of the test item. For the co-elution control, buffer solution was used in place of the lysine stock solution.

Treatment
500 µM cysteine and lysine peptide solutions were incubated in glass autosampler vials with 5 mM or 25 mM of the test item, respectively. The reaction solutions were incubated in the dark at 22.5 - 30ºC for 24 ± 2 hours prior to initiation of the analysis run. The test item and the positive control were analysed in triplicate for both peptides. The appearance of the test item and positive control samples in the HPLC vials was visually inspected and documented after preparation and prior to initiation of the HPLC run.

For detection of the cysteine and lysine peptide, the HPLC method with the following parameters was used:
Pre-column: Security Guard C18; 4,0 x 2,0 mm ID
Column: Zorbax SB C18; 2,1 x 100 mm; 3,5 µm
Eluent A: 0.1 % trifluoroacetic acid (TFA) in water
Eluent B: 0.085 % trifluoroacetic acid (TFA) in acetonitrile (ACN)

Gradient programme:
Time [min] % A % B
0.0 90 10
10.0 75 25
11.0 10 90
15.0 10 90
15.5 90 10
26.0 90 10

Flow rate: 0.35 mL/min
Detector: DAD (diode-array detector)
Wave length: 220 nm und 258 nm
Oven temperature: 30 °C
Injection volume: 2 µL
Run time: 26 min

Positive control results:

Cinnamaldehyde
Cystein depletion: 71.8 %
Lysine depletion: 44.4 %

Key result

Run / experiment:

other: Cysteine Run

Parameter:

other: Depletion in %

Value:

2.99

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Lysine run

Parameter:

other: Depletion in %

Value:

0.979

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

There were no co-elution peaks in either the cysteine or lysine assays.
The test item was analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. With an overall depletion value of 1.98%, this places the test item in the reactivity class of “no to minimal” and hence it is predicted by DPRA not to be a skin sensitizer.
A precipitate was observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item. No statement can be made about how much test item remained in the solution to react with the peptide. Since this precipitation was also observed after the incubation period of 24 ± 2 hours, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence.

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, due to the precipitation immediately upon addition of the test item solution to the peptide solution and after the incubation period of 24 ± 2 hours, the negative result of the test material cannot be used in an assessment of skin sensitisation potential.

Executive summary:

Purpose

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of the test material.

Methods

The study was performed according to OECD TG 442C.

Results

There were no co-elution peaks in either the cysteine or lysine assays.

The test item was analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. With an overall depletion value of 1.98%, this places the test item in the reactivity class of “no to minimal” and hence it is predicted by DPRA not to be a skin sensitizer.

A precipitate was observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item. No statement can be made about how much test item remained in the solution to react with the peptide. Since this precipitation was also observed after the incubation period of 24 ± 2 hours, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence.

Conclusion

In conclusion, due to the precipitation immediately upon addition of the test item solution to the peptide solution and after the incubation period of 24 ± 2 hours, the negative result of the test material cannot be used in an assessment of skin sensitisation potential.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 Feb - 20 Apr, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals: OECD 442E

Version / remarks:

June 2018

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

THP-1 Cell Cultures
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH (aliquots of cells in freezing medium at 1 × 106 to 2 × 106 cells/mL) allowing the repeated use of the same working cell stock in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured at least twice a week. The cell density should not exceed 1 × 106 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30).
The passage numbers of the used THP-1 cells were 22 and 23 in the cytotoxicity tests and 25 and 26 in the h-CLAT for runs 1 and 2, respectively.


Culture Medium
RPMI 1640 Medium, GlutaMAXTM Supplement including 25 mM HEPES, supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2 - 8 °C and used within one month.


Preparation and Seeding of THP-1 Cells
On the day of the cytotoxicity or main experiment (h-CLAT) directly before the treatment of the cells, a volume of 500 μL with a cell density of 1.8 - 2 × 106 THP-1 cells/mL was seeded in each corresponding well of a 24-well flat bottom plate.

Dose Finding Assay (Flow cytometer)
The test item concentrations investigated in the main experiment (h-CLAT) were determined with two cytotoxicity tests. The tests were performed with independent cell cultures (cells are collected from different culture flasks). The test item was prepared separately for each run. To prepare the test item, sonication of the formulation for 5 minutes and heating up to 37 °C was performed.

Treatment of the Cells
The test item dilutions were prepared freshly before each experiment.
Each DMSO solution was diluted with culture medium before application of the test solution to the cells to reach a final concentration of 0.2% (v/v) in the culture medium.
Each volume (500 μL) of the dilutions of the test item, culture medium and solvent control (0.2% (v/v) DMSO in culture medium) was added to the cells.
The treated THP-1 cells were incubated for 24 ± 0.5 hours. All dose groups were tested in one replicate for each cytotoxicity test. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, culture medium and solvent control were prepared for the 7-AAD staining.

Staining of the Cells
Each test item-treated and not test item-treated cells were collected in sample tubes centrifuged (approx. 250 × g, 5 min), washed twice (2 - 8 °C) with 2 mL FACS buffer (PBS with 0.1% (w/v) BSA) and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added in each sample tube.

Flow Cytometry Acquisition (Cytotoxicity Test)
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The cytotoxicity was analysed by flow cytometry using the software Cellquest Pro 6.0. The 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

Preparation of the acquisition (Cytotoxicity Test)
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of the FL-3 channel
The voltage of FSC and SSC was set to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot should be checked to make sure that a single population appears without contamination or excessive debris. The FL-3 voltage was set and compensate to appropriate position (FACSCalibur, Becton Dickinson GmbH, software FACSComp 6.0).
The cell viability was measured by gating-out dead cells stained with 7-AAD. A total of 10,000 living cells were analysed.
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow in the flow line.

Calculation of the Test Doses for the Main Experiment (h-CLAT)
Since the CV75 could not be determined, a stock solution of the highest soluble test item concentration was prepared and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.

Experimental Design and Procedures of h-CLAT
The test item was tested in two independent runs. The tests were performed with independent cell cultures (cells are collected from different culture flasks). The test item was prepared separately for each run.

Treatment of the Cells
For the test item exposure the highest soluble test item concentration of the cytotoxicity test (without precipitations) was used instead of 1.2 × CV75, since no CV75 could be determined. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. Each solution was diluted with culture medium before application of the test solution to the cells to reach a final concentration of 0.2% (v/v) DMSO in the medium.
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. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control was prepared 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 procedures.
The cells with the different antibodies or the IgG1 were mixed 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-AAD solution were added.


Flow Cytometry Acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

Preparation of the acquisition
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH).
The cell viability was detected by setting an R1-gate (dead cells are gated-out by staining with 7-AAD). Therefore, the R1 gate was set approximately at the middle position between the peak of the negative fraction and the positive fraction in the FL-3 histogram using DNCB-treated cells. The negative fraction corresponds to the living cells and was kept for the subsequent analyses. For each control and all test item concentrations, the cell viability was recorded from the isotype control cell tube (stained with FITC labelled-mouse IgG1), the CD54 and CD86 cell tube, where only the isotype control cells were used for the cell viability evaluation.
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.

Acquisition
Dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was calculated, but excluded from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).

Acceptance Criteria
The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control and DMSO control should be more than 90%.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
• For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability is > 90% (OECD 442E guideline).


Prediction model
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE (see chapter 5.6.7.1).
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).

Positive control results:

DNBC:
Conc. RFI RFI
[µg/mL] CD54 CD86 Viability [%]
3.0 400.8 550.4 89.55
4.0 513.4 532.7 87.86

Run / experiment:

other: Run 2 CD86 AB RFI / [%]

Parameter:

other: CD86 AB RFI / [%]

Value:

91.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Run 1 CD86 AB RFI / [%]

Parameter:

other: CD86 AB RFI / [%]

Value:

106.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Run 1 CD54 AB RFI / [%]

Parameter:

other: CD54 AB RFI / [%]

Value:

102.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Run 2 CD54 AB RFI / [%]

Parameter:

other: CD54 AB RFI / [%]

Value:

84.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (250 µg/mL). Due to the lack of cytotoxicity, a CV75 value could not be calculated. Therefore the highest concentration of the test item without precipitation was used as the highest test concentration in the main experiments.
The following concentrations of the test item were tested in the main experiments (h-CLAT):
4.35, 5.22, 6.27, 7.52, 9.03, 10.8, 13.0, 15.6 µg/mL
The test item with a log Pow of 10.4 (estimated) was tested in 2 independent runs. The RFI of CD86 and/or CD54 was not equal or greater than 150% and 200%, respectively at any dose in both runs.
In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

Run 1

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	98.07
DMSO Control	-	100.0	100.0	98.17
Positive Control (DNCB)	3.0	400.8*	550.4*	89.55
	4.0	513.4*	532.7*	87.86
Test Item	4.35	108.7	109.7	97.38
	5.22	122.8	116.2	96.98
	6.27	102.4	112.7	97.24
	7.52	96.1	100.7	97.64
	9.03	92.9	107.5	97.12
	10.8^P	95.3	90.3	97.48
	13.0^P	100.8	103.7	96.81
	15.6^P	102.4	111.2	97.21

Run 2

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	97.69
DMSO Control	-	100.0	100.0	97.25
Positive Control (DNCB)	3.0	282.7*	532.4*	87.95
	4.0	372.0*	579.8*	86.16
Test Item	4.35	94.7	100.6	97.66
	5.22	91.3	97.1	97.86
	6.27	92.0	92.0	97.55
	7.52	81.3	90.7	97.49
	9.03	75.3	90.7	97.58
	10.8^P	81.3	86.5	97.74
	13.0^P	78.0	88.5	97.68
	15.6^P	85.3	89.1	97.52

^P    Precipitation, are excluded from the evaluation

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

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, the test item with a log Pow of 10.4 (estimated) did not activate THP-1 cells up to a concentration of 9.0 µg/mL (limited by observed precipitations) under the test conditions of this study. However, the negative result cannot be used in an assessment of skin sensitisation potential because the log Pow of the test item is > 3.5 and according to the OECD test Guideline 442E, negative results for test chemicals with a log Kow (Pow) > 3.5 should not be considered.

Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of the test materialstable suspended in DMSO when administered to THP-1 cells for 24 ± 0.5 hours.

This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

Cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (250 µg/mL). Due to the lack of cytotoxicity, a CV75 value could not be calculated. Due to observed precipitations starting from 31.3 µg/mL up to 250 µg/mL the highest test concentration for the main experiment was 15.6 µg/mL.

The following concentrations of the test item were tested in the main experiments (h-CLAT):

4.35, 5.22, 6.27, 7.52, 9.03, 10.8, 13.0, 15.6 µg/mL

The test item with a log Pow of 10.4 (estimated) was tested in 2 independent runs. The RFI of CD86 and/or CD54 was not equal or greater than 150% and 200%, respectively at any dose in both runs.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria(CD54 ≥ 200% and CD86 ≥ 150%)and the cell viability was > 50%.

In conclusion, the test item with a log Pow of 10.4 (estimated) did not activate THP-1 cells up to a concentration of 9.0 µg/mL (limited by observed precipitations) under the test conditions of this study.However, the negative result cannot be used in an assessment of skin sensitisation potential because the log Pow of the test item is > 3.5 and according to the OECD test Guideline 442E, negative results for test chemicals with a log Kow (Pow) > 3.5 should not be considered.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May 08 - Aug 03, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight : 18.7 +/- 1.2 g
- Housing: grouped per dose
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: day 1 To: day 6

Vehicle:

methyl ethyl ketone

Concentration:

2.5, 5, and 10 (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 10% in MEK

Range Finding Exp. 1
- Concentration used: 5, 10 %
- Irritation:no irritation observed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Concentration: 2.5, 5, and 10%
- Criteria used to consider a positive response: Stimulation index > 3

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Standard statistical methods have been applied for data processing.

Positive control results:

Conc. SI
0%: 1.0
5% 1.99
10% 2.47
25% 5.59

Key result

Parameter:

SI

Value:

1.8

Test group / Remarks:

Test Group: 2.5% in MEK

Key result

Parameter:

SI

Value:

2.1

Test group / Remarks:

Test Group: 5% in MEK

Key result

Parameter:

SI

Value:

2.6

Test group / Remarks:

Test Group: 10% in MEK

Cellular proliferation data / Observations:

The EC3 value could not be calculated, since all group S.I.´s are below the threshold value of 3.

CLINICAL OBSERVATIONS: No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)	SD	S.I.
MEK (Vehicle Control)	726.3	241.1	1.0
2.5 % Test Item in MEK	1305.3	659.6	1.8
5 % Test Item in MEK	1520.5	559.8	2.1
10 % Test Item in MEK	1882.3	555.5	2.6

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was not a skin sensitiser under the test conditions of this study.

Executive summary:

Objective

In the study the test item formulated in methyl ethyl ketone (MEK) was assessed for its possible skin sensitising potential.

Study Design

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10 (w/w). The highest concentration tested was the highest concentration that could technically be achieved. A control group of five mice was treated with the vehicle (methyl ethyl ketone) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

Results

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.

In this study Stimulation Indices of 1.8, 2.1, and 2.6 were determined with the test item at concentrations of 2.5, 5, and 10% in methyl ethyl ketone. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Conclusion

The test item was not a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the provided information there is no need for classification according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.