Disodium 2,2'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[3-bromo-5-butyltoluene-4-sulphonate]

Endpoint summary

Description of key information

DPRA, Michel (2019)

Under the conditions of this study, the DPRA prediction is considered as positive and the test material was considered to have high peptide reactivity.

LLNA, Oroszlány (2019)

Under the conditions of this study, the test material did not show a sensitisation potential (non-sensitiser) in the Local Lymph Node Assay. No classification/labelling is triggered according to Regulation (EC) No 1272/2008 (CLP).

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:

25 September 2018 to 01 October 2018

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))

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Recommended test system in the international OECD guidelines.

Details on the study design:

MATERIALS
- Vehicle: 1:1 (v/v) mixture of acetonitrile: milli-Q water
- Positive control: Cinnamaldehyde (purity: 99.1%)
- Test material formulation preparation: The test material was pre-weighed and stored under appropriate conditions until ready to perform the testing. It was dissolved in the vehicle at 100 mM. The formulation was a blue liquid solution and was used just after its preparation.
- Co-elution control samples: prepared by incubating the test material formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.
- Reference control samples: For each peptide, the analytical batch included reference control samples (A, B or C). These samples were prepared in triplicate at the nominal concentration of 0.500 mM. These samples were used to:
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 vehicle did not impact the percentage of peptide depletion

DESIGN OF THE DIRECT PEPTIDE REACTIVITY ASSAY
The test material was tested in one run. The run was processed as described below.

Preparation of the samples
- The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.
- Co-elution control samples preparation: For the co-elution control with cysteine peptide: 50 μL of test material formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile. For the co-elution control with lysine peptide: In parallel, 250 μL of test material formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).
- Reference control samples preparation: Reference control A and B samples: In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM. Reference control C samples: Reference control C samples were prepared for each vehicle used to dissolve the test and positive control materials. For the reference control C prepared with cysteine peptide: 50 μL of each vehicle (1:1 mixture of acetonitrile: milli-Q water or acetonitrile) was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile. For the reference control C prepared with lysine peptide: In parallel, 250 μL of each vehicle (1:1 mixture of acetonitrile: milli-Q water or acetonitrile) was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).
- Cinnamaldehyde (positive control) depletion control samples preparation: For the reactivity of cinnamaldehyde with cysteine peptide: 50 μL of cinnamaldehyde at 98.2 mM in acetonitrile was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile. For the reactivity of cinnamaldehyde with lysine peptide: In parallel, 250 μL of cinnamaldehyde at 98.2 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).
- Test material samples preparation: For the reactivity of test material with cysteine peptide: 50 μL of test material formulation was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile. For the reactivity of test material with lysine peptide: In parallel, 250 μL of test material formulation was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

Incubation of the samples
- All samples (co-elution controls, reference controls, test material and positive control samples) were then 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 400 g 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 of the samples
The study samples were assayed in batches using HPLC/UV analysis.
For each peptide, the analytical sequence included at least: one blank sample (peptide dilution buffer), one calibration curve injected at the beginning of the analytical batch, three reference control A samples, the co-elution control sample, three reference control B samples, reference control C sample (replicates 1, 2 and 3), positive control sample (replicates 1, 2 and 3), test material study samples (replicates 1, 2 and 3) and three reference control B samples.

The HPLC/UV method used for the samples analysis was as follows:
- 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
- Gradient:
0 minutes: 10 % Mobile phase A, 90 % Mobile phase B
10 minutes: 25 % Mobile phase A, 75 % Mobile phase B
11 minutes: 90 % Mobile phase A, 10 % Mobile phase B
13 minutes: 90 % Mobile phase A, 10 % Mobile phase B
13.5 minutes: 10 % Mobile phase A, 90 % Mobile phase B
20 minutes: 10 % Mobile phase A, 90 % Mobile phase B
- UV Wavelength: 220 nm
- Rinse solution: Acetonitrile
- Oven temperature: 30.0 °C
- Autosampler temperature: Nominal temperature of +25 °C
- Injection volume: 7 μL
- Retention times: Cysteine-peptide: approx. 9.8 minutes, Lysine-peptide: approx. 7.5 minutes
- Total analysis time: 20 minutes

DATA ANALYSIS AND CALCULATION
- Calculation of the percentage peptide depletion: Each appropriate peak was integrated and the peak area for calibration standards, control and test material samples were determined. Based on the concentration of standards and their peak area, a linear calibration curve was generated. Then, the concentration of peptide was determined in each sample from absorbance at 220 nm, measuring the peak area of the appropriate peaks and calculating the concentration of peptide using the linear calibration curves. Then, for each positive control and test material replicate, the percentage depletion of peptide was determined from 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 vehicle) by using the following formula:
% depletion = [ 1 – (Peptide peak area in replicate injection / mean peptide peak area in relevant reference control C samples)] x 100
Then, the mean percentage depletion of the three replicates was calculated for each peptide as well as the mean of the percentage cysteine and percentage lysine depletions. Negative depletion values were considered as "Zero" for the calculation of the mean % depletion. Standard Deviation (SD) and Coefficient of Variation (CV) were calculated.
- Evaluation of the possible co-elution of the test material with the lysine or cysteine peptides: In order to detect possible co-elution of the test materials with a peptide, chromatograms of the co-elution control samples were analysed and compared with those of the reference control C samples.

ACCEPTANCE CRITERIA
The run was considered valid if the following criteria were fully met:
- the calibration curves should have a coefficient of determination (r²) ≥ 0.99
- the mean peptide concentrations of the reference control A samples should be within ± 10 % of the nominal concentration
- the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
− for the cysteine peptide, the mean percentage depletion value should be between 60.8 and 100 % with a SD < 14.9 %
− for the lysine peptide, the mean percentage depletion value should be between 40.2 and 69.0 % with a SD < 11.6 %
- the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile must be < 15.0 %.

The test material’s results were considered valid if the following criteria were fully met:
- the mean peptide concentrations of the reference control C samples prepared in the appropriate vehicle should be within ± 10 % of the nominal concentration
- the maximum SD for the test material replicates should be < 14.9 % for the percentage cysteine depletion value and < 11.6 % for the percentage lysine depletion value

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Percentage cysteine and percentage lysine depletion

Value:

80.77 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Percentage cysteine depletion

Value:

61.53 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Percentage lysine depletion

Value:

100 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

SOLUBILITY RESULTS
- During the solubility assay, the test material was found not soluble at 100 mM in acetonitrile or in milli-Q water even after 1 minute of sonication. A solution was obtained at 100 mM with a 1:1 (v/v) mixture of acetonitrile: milli-Q water. Therefore, the vehicle retained in this study was 1:1 (v/v) mixture of acetonitrile: milli-Q water.

EVALUATION OF THE PRESENCE OF PRECIPITATE AT THE END OF THE INCUBATION WITH PEPTIDES
- At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test material and positive control samples) was performed prior to HPLC analysis. As precipitate and/or phase separation (micelles) were observed in positive samples incubated with the cysteine or lysine peptides, these vials were centrifuged at 400 g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Only supernatants were then injected into the HPLC/UV system.
- For the other samples, the vials were directly transferred into the HPLC/UV system.

EVALUATION OF THE RESULTS
- 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 material did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percentage depletion values were calculated for each peptide:
- for the cysteine peptide, the mean depletion value was 61.53 %
- for the lysine peptide, the mean depletion value was 100 %
- The mean of the percentage cysteine and percentage lysine depletions was equal to 80.77 %. Accordingly, the test material was considered to have high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test material may have potential to cause skin sensitisation.

Table 1: Determination of Cysteine Peptide and Lysine Peptide Depletion in Samples Spiked with a Solution at 100 mM of Test Material

Sample Number	Cysteine Peptide		Lysine Peptide		Mean Peptide Depletion Rate (%) of Test Material	Depletion Classification
	Peak Area (µV/sec)	% Depletion	Peak Area (µV/sec)	% Depletion
1	1139767	60.65	0	100.00
2	1106449	61.80	0	100.00
3	1096788	62.14	0	100.00
Mean	-	61.53	-	100.00	80.77	High Reactivity
SD	-	0.78	-	0.00
% CV	-	1.3	-	0.0
Precipitate	No		No
Micelle	No		No

-: not applicable

Table 2: Determination of Cysteine Peptide and Lysine Peptide Depletion Concentration in Reference Control C Samples Prepared in a 1:1 Mixture of Water: Acetonitrile

Sample Number	Cysteine Peptide			Lysine Peptide
	Peak Area (µV/sec)	Concentration (mM)	%Dev	Peak Area (µV/sec)	Concentration (mM)	%Dev
1	2833902	0.497	(-0.6)	2285652	0.485	(-3.0)
2	2959573	0.519	(3.8)	2296643	0.487	(-2.5)
3	2896964	0.508	(1.6)	2286504	0.485	(-3.0)
Mean	2896813	0.508	(1.6)	2289600	0.486	(-2.8)
SD	-	0.011	-	-	0.001	-
% CV	-	2.2	-	-	0.3	-

-: not applicable

 

Table 3: Determination of Interference Due to Co-elution of the Test Material with Cysteine or Lysine Peptides

Sample Number	Peak Detected at the Cysteine Retention Time		Peak Detected at the Lysine Retention Time
	Peak Area (µV/sec)	% Interference	Peak Area (µV/sec)	% Interference
1	0	(0.0)	0	(0.0)
Precipitate	No		No
Micelle	No		No

 

Interpretation of results:

other: The DPRA prediction is considered as positive

Conclusions:

Under the conditions of this study, the DPRA prediction is considered as positive and the test material was considered to have high peptide reactivity.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442C, under GLP conditions.

The objective of this study was to evaluate the reactivity of the test material to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitisation assessment.

The reactivity of the test material was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test material and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test material 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 percentage 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 vehicle).

The test material was dissolved at 1:1 (v/v) mixture of acetonitrile: milli-Q water. 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 material did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percentage depletion values were calculated for each peptide: for the cysteine peptide, the mean depletion value was 61.53 % and for the lysine peptide, the mean depletion value was 100 %. The mean of the percentage cysteine and percentage lysine depletions was equal to 80.77 %. Accordingly, the test material was considered to have high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test material may have potential to cause skin sensitisation.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 October 2018 to 04 December 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)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

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)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old (age-matched, within one week). In the Preliminary Experiment, mice of 10 weeks of age were used.
- Weight at study initiation: 18.8 – 19.9 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.) In the Preliminary Experiment, mice of weight 18.6 – 19.6 grams were used.
- Housing: Type II. polypropylene / polycarbonate group caging. Mice were provided with glass tunnel-tubes and bedding and nesting materials were available to animals during the study.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 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: 18.1 - 24.4 °C
- Humidity: 22 - 67 %
- Air changes: 15 - 20 air exchanges/hour
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:

dimethylformamide

Concentration:

25, 10 and 5 % (w/v)

No. of animals per dose:

4 females per dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test material was examined in a short Preliminary Compatibility Test. The following standard OECD vehicles were assessed: Acetone: Olive oil 4:1 (v/v) mixture, N,N-dimethylformamide (abbreviated as DMF), Methyl ethyl ketone, Propylene glycol, Dimethyl sulfoxide, and 1 % aqueous Pluronic® PE9200. The best vehicle taking into account the test material characteristics, and the requirements of the relevant OECD guideline was considered to be DMF. 25 % (w/v) was the highest concentration which was suitable for the test. In the main assay, the test material was powdered in a mortal for a better solubility, and in both assays the formulations were treated with 15 minutes of ultrasound. The formulations appeared to be a solution by visual examination. The test material was weighed and formulations prepared daily on a weight:volume basis (as% (w/v)).

CONTROLS
- Negative control: N,N-Dimethylformamide
Animals assigned to the negative control group were treated with the vehicle only concurrent to the test material treated groups.
- Positive control: α-Hexylcinnamaldehyde
Animals assigned to the positive control group were treated with 25% (w/v) α-Hexylcinnamaldehyde solution (dissolved in DMF) concurrent to the test material treated groups.

DOSE SELECTION AND JUSTIFICATION
- Irritation and toxicity: The Preliminary Irritation/Toxicity Test was started on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test material concentrations of 25 % and 10 % (w/v) in DMF. The preliminary experiment 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. The maximum concentration of test material in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 25 % (w/v) in DMF. In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. During the Preliminary Irritation / Toxicity Test, no mortality or signs of systemic toxicity were observed. Test material residues was observed on the ear of the animals from Day 1 to Day 6 in all animals. No marked body weight loss (> 5 % reduction of body weight) was recorded for any of the animals in the assay.
- Ear thickness measurements: Ear thickness was measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. The ear thickness values and ear punch weights were within the acceptable range.
- Erythema scores: Both ears of each mouse were observed for erythema and scored. There were no indications of any irritancy at the site of application.
- The draining auricular lymph nodes of the animals were visually examined: they were normal in both groups (subjective judgement by analogy with observations of former experiments).
- Based on these observations, 25 % (w/v) dose was selected as top dose for the main test.


MAIN STUDY
- During the study, 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.
- Clinical Observations: During the study (Day 1 to Day 6) each animal was observed daily 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.
- Measurement of Body Weight: Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

PROLIFERATION ASSAY
- Injection of Tritiated Thymidine (3HTdR): On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
- Removal and Preparation of Draining Auricular Lymph Nodes: Five hours (± 30 minutes) after intravenous injection the mice were euthanised by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses). The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
- Preparation of Single Cell Suspension of Lymph Node Cells: A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
- Determination of Incorporated 3HTdR: After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4 °C), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement). The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.


EVALUATION OF THE RESULTS
- DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” ( DPM divided by the number of lymph nodes) following the industry standard for data presentation.
- Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
- Interpretation of Results: The test material is regarded as a sensitiser if both of the following criteria are fulfilled:
That exposure to at least one concentration of the test material resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
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.
- Acceptability of the test: The Local Lymph Node Assay is considered valid if it meets the following criteria:
The DPN value of the negative (vehicle) control group falls within the range of historical laboratory control data,
The positive control substance produces a significant lymphoproliferative response increases (SI > 3),
Each treated and control group includes at least 4 animals,
The test material does not cause serious systemic or local toxicity.

Positive control substance(s):

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

Positive control results:

The SI value for the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in DMF was 12.3 therefore demonstrating the appropriate performance of the assay. The lymphoproliferative response of the HCA was in line with historical control data for the positive control, therefore confirming the validity of the assay.

Key result

Parameter:

SI

Value:

2.1

Test group / Remarks:

Test Material 25 % (w/v)

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

Test Material 10 % (w/v)

Key result

Parameter:

SI

Value:

2.4

Test group / Remarks:

Test Material 5 % (w/v)

Cellular proliferation data / Observations:

CLINICAL OBSERVATION
- No mortality or signs of systemic toxicity were observed during the study. Test material residue or minimal amount of test material residue was present from Day 1 up to Day 6 in all animals of the 25, 10, and 5 % (w/v) dose groups.

BODY WEIGHT MEASUREMENT
- No marked body weight losses (≥ 5 %) were observed in any groups.

PROLIFERATION ASSAY
- The appearance of the lymph nodes was normal in the negative control group and in the test material treated dose groups. Larger than normal lymph nodes were observed in the positive control group.
- The stimulation index values were 2.1, 1.5 and 2.4 at concentrations of 25, 10, and 5 % (w/v), respectively.

INTERPRETATION OF OBSERVATIONS
- The test material was a powder, which was formulated in DMF. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test material to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these exaggerated test conditions was considered to be good evidence that the test material is a non-sensitiser. The size of lymph nodes was in good correlation with this conclusion. Based on the observed results, the test material does not need classification for skin sensitisation according to the GHS or CLP.

RELIABILITY OF THE TEST
- The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % (w/v) in the relevant vehicle (DMF) using CBA/CaOlaHsd mice.
- No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 12.3) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
- Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range. Each treated and control group included 4 animals.

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

Test Group	Measured DPM/ Group	DPM	Number of Lymph Nodes	DPN	Stimulation Index
Background (5 % (w/v) TCA)	48.5	-	-	-	-
Negative control (DMF)	2508	2459.5	8	307.4	1.0
Test Material 25 % (w/v)	5094	5045.5	8	630.7	2.1
Test Material 10 % (w/v)	3817	3768.5	8	471.1	1.5
Test Material 5 % (w/v)	5867	5818.5	8	727.3	2.4
Positive control (25% (w/v) HCA in DMF)	30269	30220.5	8	3777.6	12.3

Interpretation of results:

other: Not classified in accordance with EU Criteria

Conclusions:

Under the conditions of this study, the test material did not show a sensitisation potential (non-sensitiser) in the Local Lymph Node Assay. No classification/labelling is triggered according to Regulation (EC) No 1272/2008 (CLP).

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 429 and EU Method B42, under GLP conditions.

The aim of this study was to determine the skin sensitisation potential of the test material following dermal exposure in mice. The study was performed with vertebrate animals as classification by use of in vitro alternatives was not possible for this test material. The minimum number of animals was used, corresponding to the regulatory guidelines being followed.

The solubility of the test material was examined in a short Preliminary Compatibility Test. The following standard OECD vehicles were assessed: Acetone: Olive oil 4:1 (v/v) mixture, N,N-dimethylformamide (abbreviated as DMF), Methyl ethyl ketone, Propylene glycol, Dimethyl sulfoxide, and 1% aqueous Pluronic® PE9200. The best vehicle taking into account the test material characteristics, and the requirements of the relevant OECD guideline was considered to be DMF. 25 % (w/v) was the highest concentration which was suitable for the test. The formulations appeared to be a solution by visual examination.

A Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 25 % and 10 % (w/v) in DMF and based on the results, 25 % (w/v) was selected as top dose for the main test.

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups, each group comprised four animals: groups (three) of animals received the test material (formulated in DMF) at either 25, 10 or 5 % (w/v), a negative control group received the vehicle (DMF) only and a positive control group received 25 % (w/v) HCA (dissolved in DMF). The test material solutions were applied to the dorsal surface of the ears of the experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3) and then maintained on study for an additional 3 days. Cell proliferation in the (local) lymph nodes was assessed by measuring disintegrations per minute after the incorporation of tritiated methyl thymidine (3HTdR) into the lymph nodes and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

There was no mortality or signs of systemic toxicity observed during the study. No test material related marked body weight losses (≥ 5 %) was observed on the mean body weight changes.

The SI values were 2.1, 1.5 and 2.4 at concentrations 25, 10, and 5% (w/v), respectively.

The SI value for the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in DMF was 12.3 therefore demonstrating the appropriate performance of the assay. The lymphoproliferative response of the HCA was in line with historical control data for the positive control, therefore confirming the validity of the assay.

Under the conditions of this study, the test material did not show a sensitisation potential (non-sensitiser) in the Local Lymph Node Assay. No classification/labelling is triggered according to Regulation (EC) No 1272/2008 (CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DPRA, Michel (2019)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442C, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to evaluate the reactivity of the test material to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitisation assessment.

The reactivity of the test material was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test material and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test material 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 percentage 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 vehicle).

The test material was dissolved at 1:1 (v/v) mixture of acetonitrile: milli-Q water. 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 material did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percentage depletion values were calculated for each peptide: for the cysteine peptide, the mean depletion value was 61.53 % and for the lysine peptide, the mean depletion value was 100 %. The mean of the percentage cysteine and percentage lysine depletions was equal to 80.77 %. Accordingly, the test material was considered to have high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test material may have potential to cause skin sensitisation.

LLNA, Oroszlány (2019)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 429 and EU Method B42, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The aim of this study was to determine the skin sensitisation potential of the test material following dermal exposure in mice. The study was performed with vertebrate animals as classification by use of in vitro alternatives was not possible for this test material. The minimum number of animals was used, corresponding to the regulatory guidelines being followed.

The solubility of the test material was examined in a short Preliminary Compatibility Test. The following standard OECD vehicles were assessed: Acetone: Olive oil 4:1 (v/v) mixture, N,N-dimethylformamide (abbreviated as DMF), Methyl ethyl ketone, Propylene glycol, Dimethyl sulfoxide, and 1% aqueous Pluronic® PE9200. The best vehicle taking into account the test material characteristics, and the requirements of the relevant OECD guideline was considered to be DMF. 25 % (w/v) was the highest concentration which was suitable for the test. The formulations appeared to be a solution by visual examination.

A Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 25 % and 10 % (w/v) in DMF and based on the results, 25 % (w/v) was selected as top dose for the main test.

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups, each group comprised four animals: groups (three) of animals received the test material (formulated in DMF) at either 25, 10 or 5 % (w/v), a negative control group received the vehicle (DMF) only and a positive control group received 25 % (w/v) HCA (dissolved in DMF). The test material solutions were applied to the dorsal surface of the ears of the experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3) and then maintained on study for an additional 3 days. Cell proliferation in the (local) lymph nodes was assessed by measuring disintegrations per minutes after the incorporation of tritiated methyl thymidine (3HTdR) into the lymph nodes and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

There was no mortality or signs of systemic toxicity observed during the study. No test material related marked body weight losses (≥5 %) was observed on the mean body weight changes.

The SI values were 2.1, 1.5 and 2.4 at concentrations 25, 10, and 5% (w/v), respectively.

The SI value for the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in DMF was 12.3 therefore demonstrating the appropriate performance of the assay. The lymphoproliferative response of the HCA was in line with historical control data for the positive control, therefore confirming the validity of the assay.

Under the conditions of this study, the test material did not show a sensitisation potential (non-sensitiser) in the Local Lymph Node Assay. No classification/labelling is triggered according to Regulation (EC) No 1272/2008 (CLP).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin sensitisation.