1-(2,4-dichlorophenyl)ethan-1-one

Endpoint summary

Description of key information

Key study: OECD TG 442C. GLP study. The mean percent depletion values for cysteine and lysine peptides were 0.76% and 2.66% respectively, being the mean value equal to 1.71%. Accordingly, the test item was considered to have no or minimal peptide reactivity, though with limitations due to its precipitation or phase separation with the lysine peptide. The DPRA prediction is considered as negative.

Key study: OECD TG 442D. GLP study. Under the experimental conditions of this study, the test item, was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor. No statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations, in either run. Moreover, the Imax values were <1.5 (i.e. 1.25 and 1.00 in the first and second runs, respectively) and therefore, no EC1.5 was calculated.

Key study: OECD TG 442E. GLP study. Under the experimental conditions of this study, the test item was positive in the h-CLAT assay and therefore was considered to have potential for dendritic cell (DC) activation. RFI(CD86) and RFI(CD54) were above 150 and 200 respectively in two independent runs, yielding a positive result.

Key study: OECD 429. GLP study. Under test conditions, the test item, tested in a suitable vehicle, was shown to have a slight sensitisation potential (sensitizer) in the Local Lymph Node Assay. No confounding factors were observed. The stimulation index values were 3.1, 2.1 and 1.8 at concentrations of 50, 25 and 10 % (w/v), respectively. The calculated EC3 value is 47.5 % (w/v). Based on these results, the test item should be classified as a Skin Sensitizer, Category 1B.

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:

12 December 2017 - 18 December 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)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Cysteine peptide
. Peptide sequence : AC-RFAACAA-COOH
. Peptide sequence synonyms : AC-Arg-Phe-Ala-Ala-Cys-Ala-Ala-COOH
. Molecular weight : 750.88 g/mol
. Supplier : JPT Peptide Technologies GmbH
. Batch No. : 111016HS_MHeW1017
. Storage condition : at -20°C
. Description : White powder

Lysine peptide
. Peptide sequence : AC-RFAAKAA-COOH
. Peptide sequence synonyms : AC-Arg-Phe-Ala-Ala-Lys-Ala-Ala-COOH
. Molecular weight : 775.91 g/mol
. Supplier : JPT Peptide Technologies GmbH
. Batch No. : 220114HSDWW0117
. Storage condition : at -20°C
. Description : White powder

Vehicle:
Based on solubility results, the retained vehicle was acetonitrile without sonication step.

Test item formulation preparation:
It was dissolved in the selected vehicle (acetonitrile) at 100 Mm (without sonication step).

Positive control: Cinnamaldehyde.

Co-elution control samples:
In order to detect co-elution of the test item with a peptide, co-elution samples were prepared by incubating the test item formulation with each buffer used to dilute the peptides. Chromatograms of the co-elution control samples were analyzed and compared with those of the reference control C samples.

Reference control samples:
All these control samples were prepared in triplicate and at the nominal concentration of 0.500 mM in the solvent:
*reference control A: check the accuracy of the calibration curve for peptide quantification,
*reference control B: check the stability of the peptide during analysis,
*reference control C: check that the solvent did not impact the percentage of peptide depletion.

The test item was tested in one run.

INCUBATION OF THE SAMPLES:
All samples (co-elution controls, reference controls, test item and positive control samples) were incubated during 24 (± 2) hours at 25°C and protected from light before injection into the HPLC/UV system. At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation. Samples presenting precipitate or phase separation (micelles) were centrifuged at 400g for a period of 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system.

PREPARATION OF THE CALIBRATION CURVE SAMPLES
One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20% acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also
included in the standard calibration curve. The calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

HPLC/UV ANALYSIS
The study samples were assayed in batches using HPLC/UV analysis.
Analytical Column: Zorbax SB C18, 100 x 2.1 mm, 3.5 μm, (Waters). In-line filter C18, 4.0 x 2.0 mm (Phenomenex)
Mobile phase: Mobile phase A: acetonitrile + 0.085% TFA; Mobile phase B: milli-Q water + 0.1% TFA
Flow: 350 μL/minute
UV Wavelength: 220 nm

CALCULATION OF THE PERCENT PEPTIDE DEPLETION
% depletion = [1 - (Peptide peak area in replicate injection / Mean peptide peak area (3 replicates) in relevant reference control samples] x 100

Key result

Run / experiment:

other: 1

Parameter:

other: Mean percent depletion value for cysteine peptide

Value:

0.76

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: Mean percent depletion value for lysine peptide

Value:

2.66

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. The mean percent depletion values for cysteine and lysine peptides were 0.76% and 2.66% respectively. The mean of the percent cysteine and percent lysine depletions was equal to 1.71%. Accordingly, the test item was considered to have no or minimal peptide reactivity though with limitations due to its precipitation or phase separation with the lysine peptide. Therefore, the DPRA prediction is considered as negative and the test item may have no potential to cause skin sensitization.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for reference controls:
The mean peptide concentrations of the reference control A samples was within ± 10% of the nominal concentration. The CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile were 0.4% for cysteine and 1.6 for lysine (i.e. < 15.0%).

- Acceptance criteria met for positive control:
The cinnamaldehyde mean percent depletion for the cysteine peptide was 75.55 with a % CV of 0.4 (i.e. between 60.8 and 100%, with a SD < 14.9%). The cinnamaldehyde mean percent depletion for the lysine peptide was 62.76 with a % CV of 7.5 (i.e. between 60.8 and 100%, with a SD < 14.9%).

- Acceptance criteria met for the calibration curve samples:
The calibration curves had a coefficient of determination (r2) ≥ 0.99

- Acceptance criteria met for variability between replicate measurements:
The mean peptide concentrations of the reference control C samples prepared in acetonitrile was within ± 10% of the nominal concentration. The maximum SD for the test item replicates was < 14.9% for the percent cysteine depletion value and < 11.6% for the percent lysine depletion value.

OTHER: PRECIPITATION:
At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test item and positive control samples) was performed prior to HPLC analysis. As precipitate and phase separation (micelles) were observed in the test item samples incubated with the lysine peptides and as phase separation (micelles) were observed in co-elution samples prepared with the lysine dilution buffer, these vials were centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Positive samples incubated with both peptides were also centrifuged at the same conditions to force precipitate to the bottom of the vials. Only supernatants were then injected into the HPLC/UV system. For the other samples (i.e. all reference controls, test item and co-elution controls incubated with the cysteine peptide), the vials were directly transferred into the HPLC/UV system.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to have no or minimal peptide reactivity, though with limitations due to its precipitation or phase separation with the lysine peptide. The DPRA prediction is considered as negative.

Executive summary:

In chemico skin sensitization direct peptide reactivity assay (DPRA) was performed accordin to the OECD Guideline 442C (GLP study). The reactivity of the test item was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm. Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent). The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid. Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. The mean percent depletion values for cysteine and lysine peptides were 0.76% and 2.66% respectively, being the mean value equal to 1.71%. Accordingly, the test item was considered to have no or minimal peptide reactivity though with limitations due to its precipitation or phase separation with the lysine peptide. Therefore, the DPRA prediction is considered as negative and the test item may have no potential to cause skin sensitization.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 January 2018 - 26 January 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

VEHICLE:
Based on solubility results, the selected vehicle was DMSO.

TEST ITEM FORMULATION:
The test item was dissolved in DMSO at 200 mM. One formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2 for the 2 first runs to obtain a total of 12 concentrations in a 96-well plate; this 96-well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level. All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

SOLVENT CONTROL:
DMSO was applied to cells at a concentration of 1% in culture medium.

POSITIVE CONTROL:
Cinnamic Aldehyde (CA). For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of 2, to obtain a total of five concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 μM. All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.

TEST SYSTEM:
KeratinoSens cells: the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and
clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of luciferase gene is the endpoint evaluated and reflects the activation by the test item of the Nrf2 transcription factor in this test.
Supplier: this cell line was provided by Givaudan.
Batch: D1.
Storage condition: at -80°C.
Mycoplasm: absence of mycoplasm was confirmed.

KERATINOSENS ASSAY
Seeding:
Cells were grown using general culture procedures up to 80-90% confluence. The day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in Maintenance medium and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 104 cells/mL. Cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 μL (representing 1 x 104 cells) per well taking care to avoid sedimentation of the cells during seeding. After seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates prior to test item addition.

Treatment:
After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 μL of treatment medium. From the Master plate 4x, a volume of 50 μL was added to each well of the three white assay plates and 50 μL to the transparent plate for the cytotoxicity evaluation. All plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells. The plates were then incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.

Endpoint measurement:
After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.
Luminescence flash signal to evaluate induction signal - white plates:
After incubation, the supernatants from the white assay plates were discarded. The cells were washed once with D-PBS, a volume of 20 μL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) minutes at room temperature and under orbital shaking. The plates containing the passive lysis buffer were then placed in the luminometer for reading.
Absorbance signal to evaluate the cytotoxicity - transparent plate:
For the cell viability assay plate, the medium was replaced by 200 μL of treatment medium. A volume of 27 μL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate. The plates were covered with a sealing membrane and returned at 37°C in the icubator in humidified atmosphere for 4 hours (± 10 minutes). At the end of the incubation period, the medium was removed and a volume of 200 μL of a 10% SDS solution was added to each well. The plates were covered with a sealing membrane and placed at 37°C in the incubator in humidified
atmosphere for an overnight period to extract the formazan from cells. After the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

1.25

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

1

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Both runs were performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO. At these tested concentrations:
- No precipitate/emulsion was observed in any wells at the end of the 48-hour treatment period in either run.
- No noteworthy decrease in cell viability was noted in either run (i.e. cell viability > 70%), therefore no geometric mean IC30 or IC50 was calculated.
- No statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations, in either run. Moreover, the Imax values were <1.5 (i.e. 1.25 and 1.00 in the first and second runs, respectively) and therefore, no EC1.5 was
calculated.
The evaluation criteria for a negative response are met in both runs, the final outcome is therefore negative.

OTHER EFFECTS:
- Visible damage on test system: No.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates (11.28 and 12.35) was < 20%.
- Acceptance criteria met for positive control:
The gene induction was statistically significant above the threshold of 1.5.
The average EC1.5 value for the positive control (5.44) was within two standard deviations of the historical mean (8.4).
The average induction (Imax) in the three replicate plates for the positive control at 64 μM (6.6) was between 2 and 8.
- Range of historical values if different from the ones specified in the test guideline:
Positive contro:
EC1.5: Mean = 8.4 (n = 32); SD = 2.8; Min. = 3.8; Max. = 14.8
Imax: Mean = 8.2 (n = 32); SD = 5.4; Min. = 2.7; Max. = 22.7
Negative control:
Mean RLU: Mean = 456682.9 (n = 32); SD = 209768.4; Min. = 171403.0; Max. = 983733.0
%CV: Mean = 14.9 (n = 32); SD = 4.1; Min. = 8.3; Max. = 225.7

Evaluation of the viability (%) of cultures treated with the test item:

	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Run 1	95	105	112	106	113	112	109	110	104	102	109	100
Run 2	99	101	103	107	105	106	103	102	98	98	95	93
Mean	97	103	107	107	109	109	106	106	101	100	102	97
Geom. Mean	97	103	107	107	109	109	106	106	101	100	102	97
SD	3	3	6	1	5	4	5	6	4	3	10	5

Gene induction values obtained after treatment with the test item:

	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Run 1	1.1	1.1	1.1	1.2	1.2	0.9	1.0	1.0	1.0	1.0	1.1	0.9
Run 2	0.8	0.8	0.7	0.8	0.8	1.0	1.0	1.0	0.9	0.9	0.9	0.8
Mean	1.0	0.9	0.9	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	0.8
SD	0.2	0.2	0.3	0.3	0.3	0.0	0.0	0.0	0.1	0.0	0.2	0.1

Imax and EC1.5 results:

	Imax	EC1.5 (µM)	IC50 (µM)	IC30 (µM)
Run 1	1.25	-	-	-
Run 2	1.00	-	-	-
Mean	1.13	n.r.	n.r.	n.r.
Geom. Mean	n.r.	-	-	-
SD	0.17	-	-	-

Evaluation of the viability (%) with the positive control:

	4	8	16	32	64
Run 1	107	110	127	140	144
Run 2	105	111	125	128	130
Mean	106	110	126	134	137
Geom. Mean	106	110	126	134	137
SD	2	1	2	8	9

Imax, IC30, IC50 and EC1.5 obtained with the positive control (Induction values for the positive control):

	4	8	16	32	64	Imax	EC1.5 (µM)	IC50 (µM)	IC30 (µM)
Run 1	1.6	1.8	2.3	3.3	6.1	6.11	3.77	-	-
Run 2	1.3	1.5	1.9	3.0	7.1	7.09	7.85	-	-
Mean	1.4	1.7	2.1	3.1	6.6	6.60	n.r.	n.r.	n.r.
Geom.Mean	n.r.	n.r.	n.r.	n.r.	n.r.	n.r.	5.44	-	-
SD	0.2	0.2	0.3	0.2	0.7	0.69	2.89	-	-

Luminiscence values for the negative control wells and the %CV:

	Luminiscence reading							Mean	%CV
Run 1	Replicate 1	365138	428288	497962	506711	492266	515027	475314	11.28
	Replicate 2	438912	382266	490812	449351	436237	524442
	Replicate 3	437563	568127	499440	468245	538706	516150
Run 2	Replicate 1	588130	644098	588542	584412	684054	667825	548203	12.35
	Replicate 2	520668	494261	548345	492746	501954	525237
	Replicate 3	474241	452223	521187	537324	472512	567897

-: no data available.

n.r.: not requested by the OECD Guideline.

Interpretation of results:

GHS criteria not met

Conclusions:

Test item was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Executive summary:

An in-vitro skin sensitization Keratinosens assay was performed according to OECD Guideline 442D (GLP study). The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed. All acceptance criteria were fulfilled for the positive and negative controls in each run. Both runs were therefore considered to be valid. Both runs were performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO. At these tested concentrations no precipitate/emulsion was observed in any wells at the end of the 48-hour treatment period in either

run. No noteworthy decrease in cell viability was noted in either run (i.e. cell viability > 70%), therefore no geometric mean IC30 or IC50 was calculated. No statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations, in either run. Moreover, the Imax values were <1.5 (i.e. 1.25 and 1.00 in the first and second runs, respectively) and therefore, no EC1.5 was calculated. Under the experimental conditions of this study, the test item, was

negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06 April 2018 - 09 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline No. 442E: "In vitro skin sensitization: human Cell Line Activation Test (h CLAT)"

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol No. 158: human Cell Line Activation Test (h-CLAT)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

CONTROLS
- Solvent/vehicle control: Based on the results of the solubility assay, the vehicle selected was was dimethylsulfoxide (DMSO). As DMSO was the vehicle selected at completion of the solubility assay, DMSO control formulation was included as vehicle control, and consisted in DMSO dissolved at 0.2% in cRPMI.
- Positive controls: 2,4-Dinitrochlorobenzene (DNCB) (Sigma-Aldrich; CAS No. 97-00-7; Purity ≥ 99%) and Nickel Sulfate (NiSO4) (Merck; CAS No. 10101-97-0, Purity ≥ 99%). As several test items were assayed concurrently, the positive controls were shared. Both positive control stock solutions were prepared within 4 hours before use, and kept at room temperature and protected from light until use.
1) DNCB was prepared at the concentration of 8 µg/mL in DMSO: on the treatment day,DNCB was mixed with DMSO at a concentration of 2 mg/mL and this solution was then 250-fold diluted in cRPMI in order to obtain a 8 µg/mL stock solution.
2) NiSO4 was prepared at the concentration of 200 µg/mL in 0.9% NaCl: on the treatment day, NiSO4 was mixed with 0.9% NaCl at a concentration of 10 mg/mL and this solution was then 50-fold diluted in cRPMI in order to obtain a 200 µg/mL stock solution.

TEST SYSTEM
- Cell line used: THP-1 (immortalized human monocytic leukemia cell line derived from an acute monocytic leukemia patient).
- Source: ATCC (Ref: TIB-202, American type culture Collection, Manassas, USA), obtained by the intermediate of LGC Standards (Molsheim, France).
- Culture medium and conditions: cRPMI medium (RPMI 1640 with 10% FBS, 0.05 mM 2-mercaptoehanol and with penicillin and streptomycin). The cells were grown using general culture procedures and maintained in a humidified incubator set at 37ºC, 5% CO2 and were not allowed to exceed a cell density of 1 E06 cells/mL or more than 30 passages. During cell culturing, cell viability was checked using trypan blue.
- Reactivity check: Two weeks after thawing, a reactivity check was performed to qualify the cells before testing. The cell response after contact with Lactic Acid, DNCB and NiSO4. Results from reactivity check tests are compiled in CiToxLAB France internal data, with negative and positive control data obtained during each test.
- Cell culture for testing: Cells were seeded at a density between 0.1 E06 - 0.2 E06 cells/mL, and pre-cultured in culture flasks for 48 to 72 hours, respectively. The cell density did not exceed 1 E06 cells/mL. On the day testing, cells harvested from culture flasks were re-suspended with fresh culture medium at 2 E06 cells/mL. Then, 500 µL of cells suspension were distributed into a 24-well flat bottom plate (i. e. 1 E06 cells/well).

STUDY DESIGN
- Dose Finding assay (PI Assay). Two separate assays were conducted to assess the test item toxicity (CV75), as follows:
1) Working solutions: Test item stock solutions were prepared at 8 different concentrations by 2-fold dilutions using the selected vehicle. These formulations were then diluted 250-fold (as DMSO is the selected vehicle) into cRPMI to obtain working solutions. The final concentrations used in the assays were 7.81, 15.63, 31.25, 62.50, 125, 250, 500 and 100 µg/mL.
2) Assay: 500 µL of the working solutions were added to the volume of THP-1 cell suspension in the plate (500 µL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate. The plates were incubated for 24 h ± 30 min at 37ºC and 5% CO2. At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation. The supernatants were discarded and the cells were re-suspended with 600 µL of FACS buffer (D-PBS with 0.1% (w/v) BSA) and the plate was put into the plate-reader of a flow cytometer. A volume of Propidium Iodine (PI) solution at 12.5 µg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 µ/mL per well.

- Main test (CD86/CD54 expression measurement). Two independent validated runs were conducted as follows:
1) Working solutions: Test item formulations were prepared at 8 different concentrations by 1.2-fold dilutions in the selected vehicle. The highest concentration corresponded to 1.2-fold the mean CV75. All stock formulations were then 250-fold diluted into cRPMI to obtain working solutions. The final concentrations in the wells were (both runs): 43.47, 52.16, 62.60, 75.12, 90.14, 108.17, 129.80, 155.76 µg/mL.
2) Assay: The exposure was carried out as in the Dose Finding assay. At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation, washed twice with 1 mL of FACS buffer and blocked with 600 µL of blocking solution (0.01% globulin in FACS buffer), and incubated at 4ºC for 15 ± 1 min. After blocking, cells were split in three aliquots of 180 µL into a 96-well round bottom plate and centrifuged before staining with antibodies. A volume of 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies prepared in FACS buffer was added to each aliquot before incubation for 30 ± 2 min at 4ºC. Finally, cells were washed twice with 150 µL FACS buffer and re-suspended in 200 µL FACS buffer. The plate was then positioned into the plate-reader of the flow cytometer. A volume of 10 µL of PI solution at 12.5 µg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 µg/mL per well.
3) Flow cytometry analysis. After setting the aquisition channels for optimal detection (DRF), the non-specific binding of IgG1 and CD86 and CD54 expressionwere analyzed by flow cytometry with the acquisition channel B1 in order to obtain the Mean Fluorescence Intensity (MFI); whereas the viability (PI uptake) was analyzed with the acquisition channel B3. A total of 10 000 living cells (PI negative) were acquired. When the viability was low and did not allow obtaining 10 000 living cells, a total of 30 000 events was acquired. Alternatively, cells were acquired for a maximum of 1 min after the initiation of the acquisition. In case cell viability is less than 50%, no MFI is presented in the study report and the corresponding test item concentration are considered too high for interpretation because of the diffuse labelling cytoplasmic structures that are generated following cell membrane destruction.

CALCULATIONS
1) Estimation of the CV75 value: CV75 is defined as the estimated concentration that is required to elicit 75% cell viability. The CV75 value is derived from the dose-response curve by log-linear interpolation, using the following equation: Log CV75 = [(75 - c) x Log b - (75 - a) x Log d] / (a -c)
2) The Relative Fluorescence Intensity (RFI) of CD86 and CD54 is calculated based on the Mean Fluorescence Intensity (MFI) according to the following equation: RFI = [(MFI of test item-treated (CD86 or CD54) - MFI of test item-treated IgG1) / ((MFI of control-treated (CD86 or CD54) - MFI of control-treated IgG1)] x 100

ACCEPTANCE CRITERIA
1) Controls acceptance criteria:
- Viability of cells treated with cRPMI and DMSO controls should be ≥ 90%,
- in cRPMI and DMSO control wells, MFI ratio of both CD86 and CD54 to isotype control should be > 105%,
- in the DMSO control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI > 150% and CD54 RFI ≥ 200%),
- in the positive controls (DNCB and NiSO4), RFI values of both CD86 and CD54 should meet positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be more than 50%.

2) Test item acceptance criteria:
- For a test item noted as cytotoxic in the DRF phase, and resulting in a negative outcome in the main test, cell viability at 1.2 x CV75 should be < 90% in each run,
- cell viability of at least 4 out of 8 concentrations should be > 50%.

MAIN TEST INTERPRETATION
A run conclusion is positive if at least one of the conditions below is met:
- RFI of CD86 is ≥ 150 at any concentration leading to ≥ 50% viability,
- RFI of CD54 is ≥ 200 at any concentration leading to ≥ 50% viability.

In other circumstances, the run is considered as negative.

EVALUATION CRITERIA: Based on the individual run conclusions, a final prediction is made as follows:
- 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,
- if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE (with due consideration of the highest-tested dose conditions) 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.

Positive control results:

- DNCB: The Relative Fluorescence Intensity (RFI) of CD86 and CD54 were 426 and 590 respectively in the run A; and 226 and 544 in the run B. Because RFI(CD86) was higher than 150 and RFI(CD54) higher than 200 in both runs, the result was considered POSITIVE.
- NiSO4: The Relative Fluorescence Intensity (RFI) of CD86 and CD54 were 283 and 1521 respectively in the run A; and 174 and 2554 in the run B. Because RFI(CD86) was higher than 150 and RFI(CD54) higher than 200 in both runs, the result was considered POSITIVE.

Key result

Run / experiment:

other: A

Parameter:

other: RFI (CD86)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

DNCB = 194, NiSO4 = 207

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: B

Parameter:

other: RFI (CD86)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

DNCB = 262, NiSO4 = 241

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: A

Parameter:

other: RFI (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

DNCB = 748, NiSO4 = 1078

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: B

Parameter:

other: RFI (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

DNCB = 379, NiSO4 = 1426

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Precipitation: no abnormalities such as precipitate or cell morphology modification was observed at any tested concentrations. In the DRF assays, at post-treatment observation, emulsion was noted at concentration ≥ 125 µg/mL in both runs; in the main test, emulsion was noted in wells treated at the concentration of ≥ 129.80 µg/mL in both runs.

ACCEPTANCE OF RESULTS: All acceptance criteria were met in each run.
- Acceptance criteria met for negative control: Yes. Mean viability of cRPMI(1) and DMSO(1) control is > 90%, and for DMSO control(s): RFI(CD86) < 150 and RFI(CD54) < 200 with mean viability > 50%.
- Acceptance criteria met for positive control: Yes. DNCB and NiSO4 gives RFI(CD86) ≥ 150 and RFI(CD54) ≥ 200 with mean viability > 50%.

Dose Range Finding Results:

- DRF 1: at post-treatment observation, emulsion was noted at concentration ≥ 125 µg/mL; flow cytometry measurement after Propidium Iodide staining revealed a viability decrease below 75% at concentrations ≥ 250 µg/mL. Therefore, CV75 = 141.13 µg/mL

- DRF 2: at post-treatment observation, emulsion was noted at concentration ≥ 125 µg/mL; flow cytometry measurement after Propidium Iodide staining revealed a viability decrease below 75% at concentrations ≥ 125 µg/mL. Therefore, CV75 = 118.46 µg/mL.

- Based on the results from both runs, the mean CV75 was 129.80 µg/mL, and the highest concentration for main test was 155.76 µg/mL.

Main Test Results:

Table 2. Summary of all runs and conclusion.

Conc. (μg/mL)	RFI for CD86		RFI for CD54		Viability (%)		Run conclusion		General conclusion
	A	B	A	B	A	B	P12	P2	Positive
43.47	116	94	139	149	95.7	94.8
52.16	109	95	213	185	95.1	93.6
62.60	119	95	252	172	94.3	94.4
75.12	129	102	230	149	92.8	93.8
90.14	139	100	222	179	92.3	91.8
108.17	160	120	239	160	90.3	92.7
129.80	178	120	257	179	87.4	90.8
155.76	141	114	352	217	87.9	89.7

Table 3. Main test individual results. Run A.

Study No.		Vehicle	Run Letter	Conc. (µg/mL)	MFI (Geo Mean)			MFI ratio		IgG corrected MFI		RFI (CD86)		RFI (CD54)		Viability (%)
												Vs. Top control		Vs. Top control
					IgG	CD86	CD54	CD86 / IgG	CD54 / IgG	CD86	CD54	vs cRPMI	vs DMSO	vs cRPMI	vs DMSO	IgG	CD86	CD54	Mean
cRPMI	(3)				0.72	1.67	0.86	232	119	0.95	0.14					96.7	96.1	96.9	96.6
NiSO4	(2)	0,9% NaCl		100.00	0.70	4.07	9.81			3.37	9.11	355		6507		70.9	71.9	73.1	72.0
0,2% DMSO	(2)				0.68	1.97	0.91	290	134	1.29	0.23	136		164		95.7	96.1	95.5	95.8
DNCB	(2)	0,2% DMSO		4.00	0.74	5.51	3.19			4.77	2.45		370		1065	78.7	78.1	78.3	78.4
Test item		0,2% DMSO	A	43.47	0.75	2.24	1.07			1.49	0.32	-	116	-	139	95.7	95.9	95.7	95.7
				52.16	0.73	2.14	1.22			1.41	0.49	-	109	-	213	95.7	94.9	94.7	95.1
				62.60	0.74	2.27	1.32			1.53	0.58	-	119	-	252	94.9	94.1	94.1	94.3
				75.12	0.74	2.40	1.27			1.66	0.53	-	129	-	230	93.1	91.6	93.6	92.8
				90.14	0.73	2.52	1.24			1.79	0.51	-	139	-	222	92.6	91.3	92.9	92.3
				108.17	0.73	2.80	1.28			2.07	0.55	-	160	-	239	90.1	90.4	90.5	90.3
				129.80	0.74	3.04	1.33			2.30	0.59	-	178	-	257	87.4	88.1	86.5	87.4
				155.76	0.75	2.57	1.56			1.82	0.81	-	141	-	352	88.6	87.2	87.8	87.9

Table 4. Main test individual results. Run B.

Study No.		Vehicle	Run Letter	Conc. (µg/mL)	MFI (Geo Mean)			MFI ratio		IgG corrected MFI		RFI (CD86)		RFI (CD54)		Viability (%)
												Vs. Top control		Vs. Top control
					IgG	CD86	CD54	CD86 / IgG	CD54 / IgG	CD86	CD54	vs cRPMI	vs DMSO	vs cRPMI	vs DMSO	IgG	CD86	CD54	Mean
cRPMI	(3)				0.72	3.22	1.34	447	186	2.50	0.62					96.60	96.75	96.09	96.5
NiSO4	(2)	0,9% NaCl		100.00	0.67	7.05	13.00			6.38	12.33	255		1989		78.12	77.51	76.52	77.4
0,2% DMSO	(2)				0.71	3.69	1.18	520	166	2.98	0.47	119		76		96.59	96.16	96.08	96.3
DNCB	(2)	0,2% DMSO		4.00	0.64	6.99	3.22			6.35	2.58		213		549	80.51	80.08	80.34	80.3
Test item		0,2% DMSO	B	43.47	0.75	3.55	1.45			2.80	0.70	-	94	-	149	95.08	94.77	94.57	94.8
				52.16	0.73	3.56	1.60			2.83	0.87	-	95	-	185	93.47	93.99	93.42	93.6
				62.60	0.74	3.57	1.55			2.83	0.81	-	95	-	172	94.52	94.60	94.01	94.4
				75.12	0.76	3.80	1.46			3.04	0.70	-	102	-	149	93.94	93.56	93.97	93.8
				90.14	0.75	3.74	1.59			2.99	0.84	-	100	-	179	92.46	91.52	91.33	91.8
				108.17	0.73	4.30	1.48			3.57	0.75	-	120	-	160	93.06	92.07	93.07	92.7
				129.80	0.73	4.31	1.57			3.58	0.84	-	120	-	179	91.31	90.29	90.89	90.8
				155.76	0.72	4.11	1.74			3.39	1.02	-	114	-	217	89.09	89.51	90.42	89.7

Plate validation criteria:                                                                                         

- Mean viability of cRPMI(1) and DMSO(1) controls is > 90%                                     

- For DMSO control(s): RFI(CD86) < 150 and RFI(CD54) < 200 with mean viability > 50%

- MFI ratio for CD86 and CD54 versus IgG >105% for cRPMI(1) and DMSO(1) controls

- DNCB gives RFI(CD86) ≥ 150 and RFI(CD54) ≥ 200 with mean viability > 50%

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item was found to be positive in the h-CLAT.

Executive summary:

An in vitro cell line activation test (h-CLAT) was performed as part of an integrated approach to support the identification of the sensitization potential of the test item in accordance with the OECD Guideline 442E, following GLP. The h-CLAT method is based on changes in the quantification of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells. A solubility assay with the test item was performed and DMSO was chosen as the vehicle. Based on the results from two Dose-Range Finding assays, where the CV75 value was found to be 129.80 µg/mL, the highest concentration used in the main test was 155.76 µg/mL (i.e. 1.2-fold the mean CV75). Two validated successive test runs were performed. In each run, the test item formulations (43.47, 52.16, 62.60, 75.12, 90.14, 108.17, 129.80, 155.76 µg/mL) were applied to THP-1 cells and cultured for 24 hours and 30 minutes at 37ºC, 5% CO2. Vehicle and positive controls were run in parallel. After incubation, the expression of CD86 and CD54 was measured by cytometry analysis, and the viability of the cells was determined after being dyed with Propidium Iodine. The Mean Fluorescence Intensity (MFI) was obtained for each test sample and then corrected. The corrected MFI values were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index (RFI) for CD86 and CD54 expression. All validity criteria were met. In both runs, the results of RFI(CD86) and RFI(CD54) were above 150 and 200 respectively in all concentrations tested, both results were positive. Therefore, the test item was found to be positive in the h-CLAT.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 June 2018 - 19 June 2018

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/Ca

Remarks:

CBA/CaOlaHsd mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old
- Weight at study initiation: 19.0 – 21.0 grams. The weight variation in animals in the study did not exceed ± 20 % of the mean weight.
- Housing: Group caging / mice were provided with glass tunnel-tubes. Type II. polypropylene / polycarbo nate cages
- Diet (e.g. ad libitum): ad libitum. Ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice (Batch number: 883 29966, Expiry date: 31 October 2018; produced by ssniff Spezialdiäten GmbH, and Gel diet Transport (Batch numbers: 60180640040101 and 60181080030101, Expiry dates: 05 March 2019 and 19 April 2019, respectively) produced by Scientific Animal Food & Engineering.
- Water (e.g. ad libitum): ad libitum. tap water from municipal supply provided in 500 mL bottle.
- Acclimation period: 14 days
- Indication of any skin lesions: Only healthy animals were used for the study. Health status was certified by the veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.4 – 24.7°C
- Humidity (%): 32 – 80%
- Air changes (per hr): 15 – 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10, 25 and 50 % w/v in vehicle (based on results of preliminary test: weight changes observed at 100% w/v).

No. of animals per dose:

4 animals per dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: the best vehicle for the test item was found to be Acetone:Olive oil 4:1 (v/v) mixture (AOO). The highest achievable concentration based on the regulatory requirements of the relevant OECD guideline and the physical characteristics of the test item was 100 % (w/v).
- The Preliminary Irritation/Toxicity Test was performed in mice of 9 weeks of age (22.2-24.9 g).Two doses (2 animals/dose) at test item concentrations of 100 % (undiluted) and 50 % (w/v) in AOO were used. The test was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.
- Irritation / Erythema scores: There were no visible indications of irritancy at the site of application.
- Systemic toxicity: no mortality or signs of systemic toxicity were observed.
- Body weight: Slight body weight loss (3.0-8.0% reduction) was observed in all animals during the observation period, more significant at the highest dose.
- Ear thickness measurements: No increased ear thickness values (>25%) were detected in any of the animals at the 100 and 50% (w/v) treatment groups. The revealing ear punch weights were within the historical control range, with the exception of one animal in the 100 % (w/v) group, where slightly lower biopsy weight (12.18 mg) was measured on Day 6.

MAIN STUDY
Based on the results of pre-screen tests, 50% (w/v) was selected as top dose for the main test. Groups of four mice were treated with the test item diluted at 50%, 25% and 10% in AOO, by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

ANIMAL ASSIGNMENT AND TREATMENT
- Name of the test method: Skin Sensitization: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
a. That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
b. The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
In the main study, 3 doses of the test item and one negative and one positive control were tested. Animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Proliferation assay: On Day 6, each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS containing approximately 20 μCi of 3HTdR. Once injected, the mice were left for 5 hours (± 30 minutes). After this period mice were euthanized by asphyxiation with ascending doses of carbon dioxide. The draining auricular lymph nodes were removed and single cells suspensions of pooled lymph node cells were prepared and after overnight (approximately 18 hours) incubation at 2-8 °C, the 3HTdR incorporation was measured (10-minute measurement). The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Back ground level was also measured in duplicates.

Observations:
- Clinical observations: All animals were observed daily (Day 1 to Day 6) for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
- Body weight: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Positive control substance(s):

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

Positive control results:

The positive control substance α-Hexylcinnamaldehyde (HCA) was examined at a concentration of 25 % in the relevant vehicle (AOO) to demonstrate the appropriate performance of the assay. No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (SI value of 10.3) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay. Furthermore, the DPN values obtained were within the historical control range.

Key result

Parameter:

SI

Value:

ca. 1.4

Test group / Remarks:

10 % (w/v) in AOO

Key result

Parameter:

SI

Value:

ca. 0.9

Test group / Remarks:

25 % (w/v) in AOO

Key result

Parameter:

SI

Value:

ca. 1.2

Test group / Remarks:

50 % (w/v) in AOO

Key result

Parameter:

SI

Value:

ca. 1.1

Test group / Remarks:

100 % (w/v) in AOO

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Test item 100% (w/v) in AOO: 5451 DPM
Test item 50% (w/v) in AOO: 5859 DPM
Test item 25% (w/v) in AOO: 4311 DPM
Test item 10% (w/v) in AOO: 6733 DPM

DETAILS ON STIMULATION INDEX CALCULATION
The stimulation index values were 1.1, 1.2, 0.9 and 1.4 at a concentrations of 100 (undiluted), 50, 25 and 10% (w/v), respectively

CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity were observed during the study. Minimal amount of test item precipitate was observed on the ears of the experimental animals in the 100 % (undiluted) dose group on Days 1 3. There were no indications of any irritancy at the site of application.

BODY WEIGHTS
No treatment related effects were observed on the mean body weight of the groups. However, marked body weight loss (>5%) was detected for one animal of the negative control group and for one animal of the 25% (w/v) dose group, these changes are considered as animal variability.

OTHER: The appearance of the lymph nodes was normal in the negative control group and in all the test item treated dose groups. Larger than normal lymph nodes were observed in the positive control group.

Table 1: Main test: DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background	32	-	-	-	-
(5 % (w/v) TCA)	36
Negative control	4873	4839.0	8	604.9	1.0
(AOO)
6-Chlorohexan-2-one 100% (undiluted)	5485	5451.0	8	681.4	1.1
6-Chlorohexan-2-one 50 % (w/v) inAOO	5893	5859.0	8	732.4	1.2
6-Chlorohexan-2-one 25 % (w/v) inAOO	4345	4311.0	8	538.9	0.9
6-Chlorohexan-2-one 10 % (w/v) inAOO	6767	6733.0	8	841.6	1.4
Positive control	35008	34974.0	8	4371.8	7.2
(25 % (w/v) HCA inAOO)

Notes:

1. DPM (Disintegrations Per Minute)

2. DPN (Disintegrations Per Node) = DPM divided by the number of lymph nodes.

3. Stimulation Index = DPN of a treated group divided by DPN of the negative control group.

Interpretation of results:

GHS criteria not met

Conclusions:

Under test conditions, the test item, tested in a suitable vehicle, was shown to have a slight sensitisation potential (sensitizer) in the Local Lymph Node Assay. The stimulation index values were 3.1, 2.1 and 1.8 at concentrations of 50, 25 and 10 % (w/v), respectively. The calculated EC3 value is 47.5 % (w/v). Based on these results, the test item should be classified as a Skin Sensitizer, Category 1B.

Executive summary:

A Local Lymph Node Assay was performed to evaluate the skin sensitisation potential of the test item, in accordance with the OECD 429 Guideline, following GLP. The study was performed with vertebrate animals as classification by use of in vitro alternatives was not possible for this test item. Based on the results of preliminary solubility and irritation/toxicity tests, acetone:olive oil 4:1 (v:v) mixture (AOO) was chosen as a solvent, and the top dose for the main test was 50% (w/v), due to observations on body weight changes at 100% (w/v). In the main test, 20 female CBA/CaOlaHsd mice were used (4/group): three groups received test item at 50%, 25% and 10% (in AOO), a negative control group (vehicle, AOO) and a positive control group (25 % HCA in AOO). The test item solutions were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or systemic clinical signs were observed during the study. No test item residue was present on the ears of the animals. There were no indications of any irritancy at the site of application. No treatment related effects were observed on the mean body weight of the main study groups. Although marked body weight loss (>5% reduction) was observed in 1/4 animals in the 25 % dose group, and 2/4 animals in the 10 % (w/v) dose groups, these body weight changes were considered to be due to individual variety. The stimulation index values were 3.1, 2.1 and 1.8 at concentrations of 50, 25 and 10 % (w/v), respectively. The calculated EC3 value is 47.5 % (w/v). Based on these results, the test item should be classified as a Skin Sensitizer, Category 1B.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In chemico/in vitro studies assesing two key events of skin sensitisation were performed:

- Direct Peptide Reactivity Assay (DPRA) – B.59 / OECD TG 442C, provides information on the molecular initiation event of skin sensitization, i.e. protein binding of low molecular weight substances using synthetic heptapeptides containing cysteine and lysine amino acids. Based on the depletion of the synthetic heptapeptides by HPLC using UV detection (0.23%), the substance was determined not to be a skin sensitizer.

- ARE-Nrf2 Luciferase Test Method (KeratinoSensTM) – B.60 / OECD TG 442D, measures the induction of the luciferase gene as an indicator of the activity of the Keap1 -Nrf2 -ARE pathweay, which is considered to be a major regulator of cyto-protective responses to electrophile and oxidative stress by controlling the expression of detoxification, antioxidant and stress response enzymes and proteins. The substance was determined not to be a skin sensitizer since the luciferase induction (Imax) was lower than 1.5.

- Human Cell Line Activation Test (h-CLAT) - OECD TG 442E, provides information on dendritic cell (DC) activation by using a human monocytic leukemia cell line (THP-1) as an alternative model to DCs. Monocytic human THP1 cells used in this assay may give different signals of the same cellular molecules after stimulation with a specific substance compared to human dendritic cells. The substance was found to be positive in the h-CLAT test, since results of RFI(CD86) and RFI(CD54) were above 150 and 200 respectively in two independent runs, yielding a positive result.

- Based on a weight of evidence analysis and since inconsistent data was obtained (one out of three key events of the AOP was determined to be positive), further information was necessary to assess the skin sensitisation potential of the test item. According to point 8.3.2. of Column 2 of REACH Annex VII, an in vivo study shall be conducted if the results obtained from in vitro/in chemico test methods are not adequate for classification and risk assessment according to point 8.3.

- Local Lymph Node Assay (LLNA) - OECD TG 429. Based on the results of preliminary solubility and irritation/toxicity tests, 20 female CBA/CaOlaHsd mice (5 groups of 4 animals each) received test item at 50%, 25% and 10% in AOO, vehicle (AOO, negative control) or 25 % HCA in AOO (positive control). The test item solutions were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or systemic clinical signs were observed during the study. No test item residue was present on the ears of the animals. There were no indications of any irritancy at the site of application. No treatment related effects were observed on the mean body weight of the main study groups. Although marked body weight loss (>5% reduction) was observed in 1/4 animals in the 25 % dose group, and 2/4 animals in the 10 % (w/v) dose groups, these body weight changes were considered to be due to individual variety. The stimulation index values were 3.1, 2.1 and 1.8 at concentrations of 50, 25 and 10 % (w/v), respectively. The calculated EC3 value is 47.5 % (w/v). Based on these results, the test item should be classified as a Skin Sensitizer, Category 1B.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data (SI = 3.1, 2.1 and 1.8 at concentrations of 50, 25 and 10 % (w/v); EC3 = 47.5 % (w/v)), the substance is classified as a Skin Sensitizer, Category 1B according to the CLP Regulation (EC) No. 1272/2008.