2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

Endpoint summary

Description of key information

Skin sensitisation (in vitro)

The DPRA prediction is considered as positive and the test material may have potential to cause skin sensitisation.

Skin sensitisation (in vitro) (Michel 2017)

Under the conditions of this study, the test material was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Skin sensitisation (in vivo)

Under the conditions of the study the test material requires classification for skin sensitisation (Category 1B).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 July 2017 to 8 August 2017

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:

yes

Remarks:

(due to technical reasons the actual temperature range was 19.1-25.6°C instead of 22 ± 3°C and the actual relative humidity range was 31-80% instead of 30-70%. These deviations were considered not to adversely affect the results or integrity of the study)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old (age-matched, within one week)
- Weight at study initiation: 19.0 – 20.1 g
- Housing: Group caging in type II. polypropylene / polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days

In the Preliminary Experiment, mice of 8 weeks of age (18.3-19.3 g) were used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 - 25.6°C
- Humidity (%): 31 - 80%
- Air changes (per hr): 15 - 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Vehicle:

other: 1% aqueous Pluronic® PE9200 (1 % Pluronic)

Concentration:

Test material (formulated in 1% Pluronic) at 50, 25, and 10 % (w/v)

No. of animals per dose:

MAIN STUDY
4 animals per group (3 groups received test material at 50, 25, and 10 % (w/v). There was also a negative control group (vehicle only) and a positive control group)

Details on study design:

PRE-SCREEN TESTS:
2 animals/dose received test material at concentrations of 50 and 25 % (w/v) in 1% Pluronic. 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.
Based on the observation of the solubility test, the maximum available concentration was 50 % (w/v).
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. Both ears of each mouse were observed for erythema. Ear thickness was also 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.
Based on the observations of the preliminary test, 50 % (w/v) dose was selected as top dose for the main test.

MAIN STUDY

- Topical application
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.

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 4oC), 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.

OBSERVATIONS
- 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.
- 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.

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).
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
A test material is regarded as a sensitizer if both of the following criteria are fulfilled:
- 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.
- 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.

Since the test material gave a positive response and data permitted, the EC3 value of the test material was calculated (EC3 means the effective chemical concentration required for SI=3).

> 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 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% in the relevant vehicle (1% Pluronic) 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 3.1) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

Key result

Parameter:

SI

Value:

4.9

Test group / Remarks:

50% w/v

Key result

Parameter:

SI

Value:

3.4

Test group / Remarks:

25% w/v

Key result

Parameter:

SI

Value:

3.1

Test group / Remarks:

10% w/v

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The results of the proliferation assay are summarised in Table 1. The appearance of the lymph nodes was normal in the negative control group and in the 25 and 10% (w/v) test material treated dose groups. Larger than normal lymph nodes were observed in the 50% (w/v) test item treated group and in the positive control group.

CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity were observed during the study. Alopecia around the ears was present in the 50% (w/v) dose group from Day 3 up to Day 6. Test material precipitate or minimal amount of test material precipitate was present in all test material-treated dose groups from Day 1 up to Day 6.

BODY WEIGHTS
No marked body weight losses (≥ 5%) was observed on the mean body weight changes in any groups.

INTERPRETATION OF OBSERVATIONS
The test material was powder, which was formulated in 1% Pluronic. 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 sensitiser.

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)	32	-	-	-	-
Negative control (1% Pluronic)	1168	1136.0	8	142.0	1.0
Test material 50 % (w/v) in 1% Pluronic	5591	5559.0	8	694.9	4.9
Test mterial 25 % (w/v) in 1% Pluronic	3937	3905.0	8	488.1	3.4
Test mterial 10 % (w/v) in 1% Pluronic	3583	3551.0	8	443.9	3.1
Positive control (25 % (w/v) HCA in 1% Pluronic)	3558	3526.0	8	440.8	3.1

Interpretation of results:

other: Skin Sens. 1B in accordance with EU criteria

Conclusions:

Under the conditions of the present assay the test material, tested in a suitable vehicle, was shown to have a sensitisation potential in the Local Lymph Node Assay. The extrapolated EC3 value of Reactive Red 31 is 7.4% (w/v). The test material requires classification for skin sensitisation (category 1B).

Executive summary:

The skin sensitiation potential of the test material was investigated in a GLP study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 429.

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 50 and 25% (w/v) in 1% Pluronic. Based on the observations recorded in the preliminary test, 50% (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 of four animals each:

- three groups received test material (formulated in 1% Pluronic) at 50, 25, and 10 % (w/v) concentrations respectively,

- the negative control group received the vehicle (1% Pluronic) only,

- the positive control group received 25 % (w/v) HCA (dissolved in 1% Pluronic).

The test material 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 assessed by measuring disintegrations per minutes after incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

No mortality or signs of systemic toxicity were observed during the study. Alopecia around the ears was present in the 50% (w/v) dose group from Day 3 up to Day 6. Test material precipitate or minimal amount of test material precipitate was present in all treatment groups receiving test material formulations from Day 1 up to Day 6. No marked body weight losses (≥ 5%) was observed on the mean body weight changes in any groups.

The stimulation index values were 4.9, 3.4 and 3.1 at concentrations of 50, 25, and 10 % (w/v), respectively.

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. A lymphoproliferative response in line with historical positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

Under the conditions of the present assay the test material, tested in a suitable vehicle, was shown to have a sensitisation potential in the Local Lymph Node Assay. The extrapolated EC3 value of Reactive Red 31 is 7.4% (w/v).  The test material requires classification for skin sensitisation (category 1B).

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 June 2017 to 4 July 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:

TEST SYSTEMS
> 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_MHeW0117

> 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


PREPARATION OF 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 solvent used to dissolve the test material and positive control item.
For the reference control C prepared with cysteine peptide: 50 µL of each vehicle (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 (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 100 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 100 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 item 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 item 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 or 26 hours and 45 minutes for the lysine and cysteine analytical sequences, respectively (see § Study plan adherence) 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 were centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial 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.

DATA ANALYSIS AND CALCULATION
- alculation of the percent 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 percent 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 solvent) 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 percent depletion of the three replicates was calculated for each peptide as well as the mean of the percent cysteine and percent lysine depletions. Negative depletion values were considered as "Zero" for the calculation of the mean % depletion.
Peak areas and peptide concentrations are presented in the report. Standard Deviation (SD) and Coefficient of Variation (CV) were calculated and reported.

- 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 curve 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 percent depletion value should be between 60.8 and 100% with a SD < 14.9%,
- for the lysine peptide, the mean percent 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 solvent should be within ± 10% of the nominal concentration,
- the maximum SD for the test material replicates should be < 14.9% for the percent cysteine depletion value and < 11.6% for the percent lysine depletion value.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

0 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

100 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

other: cysteine and lysine peptides

Parameter:

other: mean depletion

Value:

50 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

SOLUBILITY RESULTS
Several vehicles were tested during the solubility assay. The test material was found not soluble at 100 mM in acetonitrile even after 1 minute of sonication. However, a solution was obtained at 100 mM in milli-Q water (without sonication step). Therefore, the vehicle retained was 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 was observed in the test material samples incubated with the cysteine peptide, 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 and reference control samples prepared in acetonitrile or in milli-Q water and incubated with the cysteine peptide 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 the lysine samples: co-elution controls, reference controls, test item and positive control 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 percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:
- for the cysteine peptide, the mean depletion value was 0.00%,
- for the lysine peptide, the mean depletion value was 100.00%.

The mean depletion value of 0.00% for the cysteine peptide could be explained by the hyperchrome effect observed on the peak area. The peak area for the test material was approximately 4 to 5-fold higher than peak area of the reference control C.
It is to be noted that since precipitate was observed at the end of the incubation with the cysteine peptide, the corresponding peptide depletion may be underestimated. Therefore, the mean depletion value of 0.00% cannot be drawn with sufficient confidence.

The mean of the percent cysteine and percent lysine depletions was equal to 50.00%. Accordingly, the test material was considered to have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test material may have potential to cause skin sensitisation.

Interpretation of results:

other: Skin sensitiser in accordance with EU criteria

Conclusions:

Under the experimental conditions of this study, the test material was considered to have a high peptide reactivity. The test material is considered positive in the DPRA assay.

Executive summary:

The skin sensitisation potential of the test material was investigated in a GLP study which was conducted in accordance with the standardised guideline OECD 442C.

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 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 test material was dissolved at 100 mM in 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 item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide:

- for the cysteine peptide, the mean depletion value was 0.00%,

- for the lysine peptide, the mean depletion value was 100.00%.

The mean depletion value of 0.00% for the cysteine peptide could be explained by the hyperchrome effect observed on the peak area. The peak area for the test material was approximately 4 to 5-fold higher than peak area of the reference control C.

It is to be noted that since precipitate was observed at the end of the incubation with the cysteine peptide, the corresponding peptide depletion may be underestimated. Therefore, the mean depletion value of 0.00% cannot be drawn with sufficient confidence.

The mean of the percent cysteine and percent lysine depletions was equal to 50.00%. Accordingly, the test material was considered to have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test material may have potential to cause skin sensitisation.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 June 2017 to 21 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 2015

Deviations:

yes

Remarks:

See below.

Principles of method if other than guideline:

DEVIATIONS
Luminescence flash signal to evaluate induction signal – white plates: During the first run, a technical issue related to the Varioskan Flash occurred during the luminescence reading of the first white plate.
As a consequence:
- No luminescence data was available for the two lower concentrations of the test material and for the first two negative control wells. Therefore, the two lower concentrations of the test material were analysed in duplicate instead of triplicate and the negative control was analysed from 16 replicates instead of 18.
- Solving this technical issue resulted in a delay. Thus, the cells were incubated with the passive lysis buffer during 62 and 57 minutes, for the first and the second white plate, respectively, instead of within 30 minutes maximum.
- However, the luminescence values of the negative control wells were similar between the three different white plates and in particular with the third white plate which was correctly incubated during 18 minutes (in agreement with the study method). In addition, the average Coefficient of Variation of the luminescence reading in the negative control wells of the triplicate plates was < 20 % (i.e. 10.82 %). Also, the induction profiles of the test material were similar between the three white plates. Thus, the signal was constant across the triplicate white plates.
These deviations were considered to not have compromised the validity or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test material was dissolved in water for injections at 200 mM. After preparation, the formulations were sterilised by filtration through a filter prior treatment. One formulation was prepared for each run.
- Preparation of the test chemical serial dilutions: This solution was diluted in water for injections by serial dilutions, using a dilution factor of 2, 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
- Preparation of the positive controls: For each run, the positive control material (Cinnamic Aldehyde (CA)) 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 5 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.
- Preparation of the solvent, vehicle and negative controls: based on solubility results, the vehicle was water for injections. The negative control was DMSO.

SOLUBILITY ASSAY:
- A solubility assay was performed prior the first treatment in order to select the vehicle (among DMSO, water for injections or treatment culture medium).

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3 assay plates.
- Number of repetitions: 2 independent runs using cells from a different passage number.
- Test chemical concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.4, 125, 250, 500, 1 000 and 2 000 μM in culture medium containing 1 % DMSO and 1 % water for injections.
- Exposure time: 48 (± 2) hours
- Study evaluation and decision criteria used: The results of each run are analysed individually and if the test material is classified as positive in two runs, the final outcome is considered positive. If the test material is classified as negative in two runs, the final outcome is negative. In case, the first two runs were not concordant, a third run was performed and the final outcome was that of the two concordant runs.
The test material is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
• The Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test)
• At the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70 %
• The EC1.5 value is < 1 000 μM (or < 200 μg/mL for test material without MW)
• There is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).
- Description on study acceptance criteria: Each run was considered valid if the following criteria were met:
• The positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations.
• The average EC1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8. If the latter criterion was not fulfilled, the dose-response of cinnamic aldehyde was carefully checked, and the run was accepted if there was a clear dose-response with increasing luciferase activity at increasing concentrations for the positive control.
• The average Coefficient of Variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20.

SEEDING AND INCUBATION
- Seeding conditions: 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 EDTA, harvested, re-suspended in Maintenance medium No. 2 and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 10^4 cells/mL
- Cells were distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 μL (representing 1 x 10^4 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 material addition. 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 materials and to avoid cross-contamination between wells.
- Incubation conditions: 48 (± 2) hours at 37 °C, 5 % CO2, 90 % humidity.
- Precipitation observation: After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.

LUCIFERASE ACTIVITY MEASUREMENTS
- Luminometer: 96-well plate Luminometer with injectors and optical density reader (Varioskan Flash).
- Lysate preparation: 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 for the third plate and for 62 or 57 minutes, for the first and second plates respectively, at room temperature and under orbital shaking,
- The following program was used for the luminometer, 50 μL of the luciferase substrate was added to each well and 1 second after this addition, the luciferase signal was integrated for 2 seconds.

DATA EVALUATION
- Cytotoxicity assessment: The absorbance signal was used to evaluate the cytotoxicity:
• 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 incubator 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.
- Prediction model used: KeratinoSens

Vehicle / solvent control:

water

Negative control:

other: DMSO applied at 1 % in culture medium.

Positive control:

cinnamic aldehyde [442D]

Positive control results:

In run 1 the Imax was 17.08 and the EC1.6 was 6.94 μM.
In run 2 the Imax was 14.66 and the EC1.6 was 6.09 μM.
The Imax mean was 15.87. The geometric mean for the EC1.6 was 6.5 μM.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC30 [442D]

Value:

1 901.8 µM

Cell viability:

A decrease in cell viability (i.e. cell viability < 70 %) was observed at 62.5, 125 and 2 000 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC30 [442D]

Value:

1 681.74 µM

Cell viability:

A decrease in cell viability (i.e. cell viability < 70 %) was noted at 2 000 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.28

Cell viability:

A decrease in cell viability (i.e. cell viability < 70 %) was observed at 62.5, 125 and 2 000 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

0.86

Cell viability:

A decrease in cell viability (i.e. cell viability < 70 %) was noted at 2 000 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

SOLUBILITY TEST
- In the solubility test, the test material was found not soluble in DMSO at 200 mM, whereas it was found soluble in water for injections at 200 mM. Therefore, this vehicle (water for injections) was selected for the preparation of the test material stock formulations.

KERATINOSENS RUN:
- Precipitation: No precipitate/emulsion was observed in any test material treated-wells at the end of the 48-hour treatment period in either run.
- Colouration: A red colouration was observed in treated wells at concentrations ≥ 15.63 μM in both runs.
- IC50: No IC50 was calculated since the cell viability was > 50 % in both runs.
- Gene-fold induction: 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.
- No EC1.5 was calculated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All acceptance criteria were fulfilled for the negative control in both runs, they were therefore considered as validated.
- Acceptance criteria met for positive control: All acceptance criteria were fulfilled for the positive control in both runs, they were therefore considered as validated. In both runs, the criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8" was not fulfilled (i.e. Imax of 17.08 or 14.66, respectively). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control in both runs, this was considered not to have any impact on the validity of the results of this study.
- The evaluation criteria for a negative response was met in both runs, the final outcome was therefore negative.

Evaluation of the viability (%) of the cultures treated with the test material for each run

	Concentrations (μM)
Test Material	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Viability (%) in Run 1	106	105	101	101	82	71	68	67	73	82	92	56
Viability (%) in Run 2	91	110	104	85	84	80	85	86	94	107	104	54
Mean viability (%)	98	107	102	93	83	75	76	77	84	94	98	55
Geometric Mean (%)	98	107	102	92	83	75	76	76	83	93	98	55
SD	10	4	2	12	2	7	12	13	15	18	8	1

Gene induction values, Imax, IC30, IC50 and EC1.5 values, mean and SD values obtained after treatment with the test material in each run

	Concentrations (μM)
Test Material	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Induction values in Run 1	0.7	0.5	0.7	0.6	0.6	0.5	0.5	0.6	1.0	1.3	1.3	0.9
Induction values in Run 2	0.7	0.8	0.7	0.5	0.4	0.4	0.4	0.4	0.6	0.9	0.7	0.8
Mean induction	0.7	0.7	0.7	0.6	0.5	0.4	0.5	0.5	0.8	1.1	1.0	0.9
SD	0.0	0.2	0.0	0.1	0.1	0.1	0.1	0.1	0.3	0.3	0.4	0.0

Imax and EC1.5 results

Test Material	Imax	EC1.5 (μM)	IC50 (μM)	IC30 (μM)
Run 1	1.28	-	-	1901.80
Run 2	0.86	-	-	1681.74
Mean	1.07	n.r.	n.r.	n.r.
Geometric Mean	n.r.	-	-	1788.39
SD	0.30	-	-	155.61

-: No data available

n.r.: Not requested by OECD Guideline

Evaluation of the viability (%) of cultures treated with the positive control for each run

	Concentrations (μM)
Cinnamic aldehyde	4	8	16	32	64
Viability (%) in Run 1	106	115	117	126	113
Viability (%) in Run 2	103	110	118	130	135
Mean viability (%)	104	113	117	128	124
Geometric Mean (%)	104	113	117	128	124
SD	2	4	0	3	16

Gene induction values, Imax, IC30, IC50 and EC1.5 values obtained with the positive control for each run

	Concentrations (μM)
Cinnamic aldehyde	4	8	16	32	64	Imax	EC1.5 (μM)	IC50 (μM)	IC30 (μM)
Run 1	1.4	1.5	2.5	3.6	17.1	17.08	6.94	-	-
Run 2	1.4	1.6	2.0	3.5	14.7	14.66	6.09	-	-
Mean	1.4	1.6	2.2	3.6	15.9	15.87	n.r.	n.r.	n.r.
Geometric Mean	n.r.	n.r.	n.r.	n.r.	n.r.	n.r.	6.50	-	-
SD	0.0	0.0	0.4	0.0	1.7	1.71	0.60	-	-

 -: No data available

n.r.: Not requested by OECD Guideline

Historical data from 13 October 2015 to 13 October 2016

Control Material	Negative Control		Positive Control
Parameter	Mean RLU	%CV	EC1.5	Imax
n	28	28	28	28
Mean	469622.2	15.3	13.9	4.7
SD	302495.1	4.2	6.6	3.3
Lower CL 95 %	352326.9	13.6	11.4	3.4
Upper Cl 95 %	586917.6	16.9	16.5	6.0
5^thPercentile	181303.0	8.3	4.1	2.8
Median	326775.0	15.0	12.1	3.7
95^thPercentile	1045640.0	22.5	22.6	15.1
Min	180496.0	8.3	2.8	2.6
Max	1093648.0	25.7	29.0	15.9
Mean – 2 SD	/	6.8	0.8	/
Mean + 2 SD	1074612.4	23.7	27.1	11.2

CL: Confidence limit

CV: Coefficient of Variation

EC1.5: Extrapolated concentration for a 1.5-fold luciferase gene induction

Imax: Maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured.

Min: Minimal value

Max: Maximal value

n: Number of values

RLU: Relative Luminescence Unit

SD: Standard Deviation

/: Not applicable, negative calculated value

Interpretation of results:

other: Negative in the KeratinoSens assay

Conclusions:

Under the conditions of this study, the test material was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Executive summary:

The skin sensitisation potential of the test material in vitro was investigated using a KeratinoSens assay in accordance with the standardised guideline OECD 203 under GLP conditions.

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 material and of positive controls (cinnamic aldehyde). 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.

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, 1 000 and 2 000 μM in culture medium containing 1 % DMSO and 1 % water for injections.

At these tested concentrations:

- No precipitate/emulsion was observed in any test material treated-wells at the end of the 48-hour treatment period in either run.

- A red colouration was observed in treated wells at concentrations ≥ 15.63 μM in both runs.

- A decrease in cell viability (i.e. cell viability < 70 %) was noted at concentrations of 62.5, 125 then 2 000 μM in the first run and at the concentration of 2 000 μM in the second run.

- The corresponding IC30 were calculated to be 1 901.80 and 1 681.74 μM in the first and second run, respectively, and no IC50 was calculated since the cell viability was > 50 % in both runs.

- 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.

- The Imax values were ≤ 1.5 in both runs (i.e. 1.28 and 0.86 in the first and second runs, respectively) and no EC1.5 was calculated.

The evaluation criteria for a negative response were met in both runs, the final outcome is therefore negative.

Under the conditions of this study, the test material was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisation (in chemico)

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

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 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 test material was dissolved at 100 mM in 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 item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide:

- for the cysteine peptide, the mean depletion value was 0.00%,

- for the lysine peptide, the mean depletion value was 100.00%.

The mean depletion value of 0.00% for the cysteine peptide could be explained by the hyperchrome effect observed on the peak area. The peak area for the test material was approximately 4 to 5-fold higher than peak area of the reference control C.

It is to be noted that since precipitate was observed at the end of the incubation with the cysteine peptide, the corresponding peptide depletion may be underestimated. Therefore, the mean depletion value of 0.00% cannot be drawn with sufficient confidence.

The mean of the percent cysteine and percent lysine depletions was equal to 50.00%. Accordingly, the test material was considered to have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test material may have potential to cause skin sensitisation.

Skin sensitisation (in vivo)

The skin sensitiation potential of the test material was investigated in a GLP study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 429. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 50 and 25% (w/v) in 1% Pluronic. Based on the observations recorded in the preliminary test, 50% (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 of four animals each:

- three groups received test material (formulated in 1% Pluronic) at 50, 25, and 10 % (w/v) concentrations respectively,

- the negative control group received the vehicle (1% Pluronic) only,

- the positive control group received 25 % (w/v) HCA (dissolved in 1% Pluronic).

The test material 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 assessed by measuring disintegrations per minutes after incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

No mortality or signs of systemic toxicity were observed during the study. Alopecia around the ears was present in the 50% (w/v) dose group from Day 3 up to Day 6. Test material precipitate or minimal amount of test material precipitate was present in all treatment groups receiving test material formulations from Day 1 up to Day 6. No marked body weight losses (≥ 5%) was observed on the mean body weight changes in any groups.

The stimulation index values were 4.9, 3.4 and 3.1 at concentrations of 50, 25, and 10 % (w/v), respectively.

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. A lymphoproliferative response in line with historical positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

Under the conditions of the present assay the test material, tested in a suitable vehicle, was shown to have a sensitisation potential in the Local Lymph Node Assay. The extrapolated EC3 value of Reactive Red 31 is 7.4% (w/v).  The test material requires classification for skin sensitisation (category 1B).

Skin sensitisation (in vitro) (Michel 2017)

The skin sensitisation potential of the test material in vitro was investigated using a KeratinoSens assay in accordance with the standardised guidelines OECD 203 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 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 material and of positive controls (cinnamic aldehyde). 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.

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, 1 000 and 2 000 μM in culture medium containing 1 % DMSO and 1 % water for injections.

At these tested concentrations:

- No precipitate/emulsion was observed in any test material treated-wells at the end of the 48-hour treatment period in either run.

- A red colouration was observed in treated wells at concentrations ≥ 15.63 μM in both runs.

- A decrease in cell viability (i.e. cell viability < 70 %) was noted at concentrations of 62.5, 125 then 2 000 μM in the first run and at the concentration of 2 000 μM in the second run.

- The corresponding IC30 were calculated to be 1 901.80 and 1 681.74 μM in the first and second run, respectively, and no IC50 was calculated since the cell viability was > 50 % in both runs.

- 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.

- The Imax values were ≤ 1.5 in both runs (i.e. 1.28 and 0.86 in the first and second runs, respectively) and no EC1.5 was calculated.

The evaluation criteria for a negative response were met in both runs, the final outcome is therefore negative.

Under the conditions of this study, the test material was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

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 require classification with respect to skin sensitisation (Category 1B).