Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

 Endpoint study report Test   Year  GLP (Y/N)  Adequacy (Y/N)  Product tested as  Relevance (Y/N)  Results Remarks  Reliability (Klimish Method) 
 skin.Sensitization.003 LLNA 2017  Y  Y

Liquid

(12.5%, 25% and 50% in 2% CMC aqueous solution)

Y  Invalid  No dose-response in these experimental conditions due to incompatibilities with the gelling agent used
 Skin sensitization.001 h-CLAT   2018  Y  Y

 Liquid

(from 827 to 2965 µg/mL in saline solution)

 Positive  

Activation of the dendritic cell with CD-86 marker on 2 independent runs.

(exclusion of the 2965 g/ml value because of cytotoxicity)

 Skin sensitization.002  KeratinoSens 2018   Y  Y

 Liquid(from 0.2 to 400 µg/ml in saline solution)

 Y  Negative  The 3 repetitions were negative under the retained experimental conditions  1
 Skin sensitization.005  ISO Closed Patch Sensitization Study in Guinea Pig  2017  Y  Y

 Liquid

(40.4 g/l in saline solution) (The tested product is X113, which is diluted X330 in saline solution)

Y  Negative -  1
 Skin sensitization.007  Clinical evaluation report 2018   N/A  Y

 Liquid

(40.4 g/L in saline solution) (The tested product is X113, which is diluted X330 in saline solution)

 Negative  Human data available on X113, especially focus on a clinical study performed on human volunteers.
 Skin sensitization.004 rLLNA 2011   N

Liquid

(161 g/L in saline solution) (The tested product is X4412, which is diluted X330 in saline solution)

Y  Negative -

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), July 2016
Deviations:
yes
Remarks:
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: #070602
- Expiration date of the lot/batch: 06/06/2019
- Purity test date: 97.49%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, moisture protected
- Solubility and stability of the test substance in the solvent/vehicle: soluble ans stable in water or saline

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dissolution in saline
- Final dilution of a dissolved solid, stock liquid or gel: 827, 993, 1192, 1430, 1716, 2059, 2471 and 2965 μg/mL

FORM AS APPLIED IN THE TEST: liquid (powder dissolved in saline)
Details on the study design:
the dendritic cell activation potential is the third key event of a skin sensitization AOP.

Culture medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin)
Solvent control: saline (0.9% NaCl), diluted 1:100 in culture medium
Medium control: culture medium
Positive control: DNCB
Concentration of the positive control: 2 and 3 µg/ml
Solvent of the positive control: DMSO 0.2% in culture medium
Solvent control of the positive control: DMSO 0.2% in culture medium

test system: THP-1 cells, from ATCC, #TIB-202
Justification for this test system: THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.
THP-1 Cell Cultures: aliquots of cells in freezing medium at 1 × 10E6 to 2 × 10E6 cells/mL
Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1 × 10E6 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere.
Prior to using a THP-1 cell batch for testing, the cells were qualified by conducting a reactivity check.
The passage numbers of the used THP-1 cells were 20 and 8 in the XTT assays and 12 and 13 in the h-CLAT for runs 1 and 2, respectively.
Preparation and Seeding of THP-1 Cells: seeded at a density between 0.2 × 10E6 cells/mL and 0.5 × 10E6 cells/mL + pre-cultured in culture flasks for 48 or 72 hours.
XTT experiments: a volume of 100 μL with a cell density of 0.9 - 1 × 10E6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate.
Main experiment: cells were resuspended at 2 × 10E6 cells/mL. For the main experiment (h-CLAT) 0.9 - 1 × 10E6 cells/well in a volume of 500 μL were seeded in a 24-well plate before the treatment.
Dose finding assay: XTT test
Number of test: 2
Acceptability of the assay:
• mean absorbance of the medium control is ≥ 0.5
• mean viability of the solvent control is ≥ 90% in comparison to the medium control
Positive control results:
The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.
Run / experiment:
other: Mean of run 1 and run 2
Parameter:
other: meanCV75
Remarks:
(µg/ml)
Value:
2 470.85
Key result
Run / experiment:
other: 1
Parameter:
other: RFI%
Remarks:
CD86
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: 2
Parameter:
other: RFI%
Remarks:
CD86
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: 1
Parameter:
other: RFI%
Remarks:
CD54
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2
Parameter:
other: RFI%
Remarks:
CD54
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
- Acceptance criteria met for positive control:
- Acceptance criteria met for variability between replicate measurements:
- Range of historical values if different from the ones specified in the test guideline:
DEMONSTRATION OF TECHNICAL PROFICIENCY:Results of the h-CLAT proficviency of the lab are provided on a list of defined chemicals as described into the guideline.

ACCEPTANCE OF RESULTS:
Cytotoxicity test: The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%

- Acceptance criteria met for negative control: RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).

- Acceptance criteria met for positive control:The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%

 Concentration (µg/ml)  CD 54 (RFI%)  CD 86 (RFI%)  % viability    CD 54 (RFI%)   CD 86 (RFI%)   % viability 
827   109.1  229.8  86.0  96.6  185.4  96.2
 993 145.5  233.3  76.2  111.2  240.9  93.3
 1192    131.8   300.9  83.8  112.4  278.1  94.6
      
 1430  133.3  369.7  83.3  94.4  297.1  92.4
 1716  139.4  451.3  80.0  96.6  325.5  92.0
 2059  151.5  348.7  70.4  111.2  410.6  83.8
 2471  183.3  562.3  63.7  123.6  479.6  69.4
 2965  190.9*  683.8*  46.2*  152.8  675.5  50.7

* cell viability below 50%, are excluded from the evaluation

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The test item X330 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).
Executive summary:

The test item X330 was tested for its potential to induce skin sensitization in a strategy of assay. It was tested according to OECD guideline 442E, In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation, with the human cell Line Activation Test or h-CLAT method.

The test item X330 was dissolved in saline and further diluted in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of X330 was previously determined by two XTT tests (deviation from the guideline but XTT tests are recognized as tests to assess the cytotoxicity).

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 2500 μg/mL up to the highest tested concentration (5000 μg/mL) in the first XTT test and with the highest tested concentration (5000 μg/mL) in the second XTT test (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 2470.85 μg/mL.

The changes of surface markers expression (CD54, CD86) are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. The relative fluorescence or luminescence intensity of the treated cells compared to solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.

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

827, 993, 1192, 1430, 1716, 2059, 2471 and 2965 μg/mL.

The test item X330 was tested in 2 independent runs. The highest tested test item concentration (2965 μg/mL) of the first h-CLAT run was excluded from the evaluation, since the cell viability was below 50%. The RFI of CD86 was greater than 150% in all concentrations of both runs. Therefore, the h-CLAT prediction is considered positive for the tested test item in this h-CLAT. A median EC150 and EC200 value could not be calculated.

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

In conclusion, the test item X330 with a log Pow of -4.9 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ECVAM DB-ALM protocol 155: Keratinosens(TM)
Principles of method if other than guideline:
TEST SYSTEM: Keratinosens™,
CELL TYPE: engineered skin cell line
SOURCE: GIVAUDAN
Microbiology test: cells are exempts of mycoplasma
STORAGE CONDITIONS: cultured in maintenance medium (DMEM 1 g/l glucose, 9,1% non-heat inactivated foetal calf serum, 0.05% geneticin) at 37°C, 5% CO2
Number of passage: 20 (runs 1 and 2) and 17 (run 3)
VEHICLE: Saline solution (0.9% NaCl).
JUSTIFICATION OF THE CHOICE OF THE VEHICLE: test item is soluble in saline solution and insoluble in DMSO.

- Luminometer: GloMax™, PROMEGA
- MULTISKAN EX plate reader (Thermo life Sciences), reading range 0 – 3.5 units of absorbance. Linearity range: 0 – 2.200 units of absorbance

Positive control: Cinnamaldehyde
Negative control: Treatment culture medium, 1% DMSO, 1% Non-heat inactivated foetal calf serum
Diluents for the test item: Sterile water - stored at room temperature 20°C ± 5°C
- Diluent for the positive control: DMSO (1% maximum final concentration) - stored at room temperature 20°C ± 5°C

- Luciferase substrate: Bright Glo™ Luciferase Assay System (Promega) - stored at -80°C after reconstitution

- Cell washing solution: Dulbecco’s PBS Ca2+ and Mg2+ free with 0.05% EDTA - stored at 5°C ± 3°C
- Dulbecco’s PBS Ca2+ and Mg2+ free - stored at room temperature 20°C ± 5°C
- Staining solution: 5 mg/ml MTT* (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution in PBS - prepared extemporaneously and used within the day
- Desorption solution: 10% SDS in water - stored at room temperature 20°C ± 5°C

TEST PROTOCOL:
CELLS SEEDING THE 1ST DAY
Cell density: 10E4 cells per well, incubated 24h at 37°C, 5% CO2 after having been trypsinized.

PREPARATION OF TEST ITEM DILUTIONS (2ND DAY)
Stock solution: 40 mg/ml
positive control preparation: 6,4 mM in DMSO

Preparation of the 100 X plate
A 100-fold concentrated dilutions series was prepared in 96-well plate (test item, positive control, negative control).

Preparation of the 4 X dilution plate
The 100 X DMSO plate was diluted 25 fold in a new plate (4 X). For the test item, the level of the DMSO is adjusted to 1% final.

Contact between the cells and the test and reference items: replacement of 150 µl treatment medium for the 5 seeded plates.
4 X plate: replicated 5 times. Volume: 50 µl
Plate 1X: covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37°C, 5% CO2).

DAY 4: LUCIFERASE ACTIVITY
After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then 100 µl of luciferase substrate (luciferine + ATP + lysing agent) were then added in each well.
New incubation time: 15 min at room temperature.
Measurement with a luminometer.

CELL VIABILITY ASSESSMENT (MTT DYE TEST):
MTT solution: diluted at 0.6 mg/ml in treatment medium
MTT volume: 225 µl
MTT incubation: 4 hours (+/-30 min) at 37°C, 5% CO2
Elimination of the staining solution and treatment with 200 µl of 10% SDS one night in the dark (37°C, 5% CO2)
Measurement of the absorbance at 540 nm.

ACCEPTABILITY CRITERIA

- Positive control:
o Imax > 1.5 in at least one dose,
o EC1.5 within two standard deviations of the historical mean : 2.9 µM ≤EC1.5≤22.6 µM and the mean induction in each run for cinnamaldehyde at 64 µM between 2 and 8.
- Control solvent:
o the average coefficient of variation of the luminescence (CV%) reading for the negative (solvent) control DMSO should be < 20% in each repetition

PREDICTION MODEL

The test item is a potential skin sensitizer if the 4 following parameters are met in 2/2 or 2/3 repetitions. Otherwise the prediction is concluded negative:
1. Imax> 1.5 and statistically significantly different as compared to the solvent control (as determined by a 2-tailed, unpaired Student’s T-test)
2. Cellular viability > 70% for Imax> 1.5 (lowest concentration)
3. EC1.5 < 200 µg/ml
4. Apparent overall dose-reponse for luciferase induction
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
LLNA test already performed was unvalid. New in vitro tests were validated in 2016.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: #070602
- Expiration date of the lot/batch: 06/06/2019
- Purity test date: 97.49%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, moisture protected

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: dissolved in saline solution and tested at 12 concentrations, from 0.2 µg/ml to 400 µg/ml.
Details on the study design:
TEST SYSTEM: Keratinosens™,
CELL TYPE: engineered skin cell line
SOURCE: GIVAUDAN
Microbiology test: cells are exempts of mycoplasma
STORAGE CONDITIONS: cultured in maintenance medium (DMEM 1 g/l glucose, 9,1% non-heat inactivated foetal calf serum, 0.05% geneticin) at 37°C, 5% CO2
Number of passage: 20 (runs 1 and 2) and 17 (run 3)
VEHICLE: Saline solution (0.9% NaCl).
JUSTIFICATION OF THE CHOICE OF THE VEHICLE: test item is soluble in saline solution and insoluble in DMSO.

- Luminometer: GloMax™, PROMEGA
- MULTISKAN EX plate reader (Thermo life Sciences), reading range 0 – 3.5 units of absorbance. Linearity range: 0 – 2.200 units of absorbance

Positive control: Cinnamaldehyde
Negative control: Treatment culture medium, 1% DMSO, 1% Non-heat inactivated foetal calf serum
Diluents for the test item: Sterile water - stored at room temperature 20°C ± 5°C
- Diluent for the positive control: DMSO (1% maximum final concentration) - stored at room temperature 20°C ± 5°C

- Luciferase substrate: Bright Glo™ Luciferase Assay System (Promega) - stored at -80°C after reconstitution

- Cell washing solution: Dulbecco’s PBS Ca2+ and Mg2+ free with 0.05% EDTA - stored at 5°C ± 3°C
- Dulbecco’s PBS Ca2+ and Mg2+ free - stored at room temperature 20°C ± 5°C
- Staining solution: 5 mg/ml MTT* (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution in PBS - prepared extemporaneously and used within the day
- Desorption solution: 10% SDS in water - stored at room temperature 20°C ± 5°C

TEST PROTOCOL:
CELLS SEEDING THE 1ST DAY
Cell density: 10E4 cells per well, incubated 24h at 37°C, 5% CO2 after having been trypsinized.

PREPARATION OF TEST ITEM DILUTIONS (2ND DAY)
Stock solution: 40 mg/ml
positive control preparation: 6,4 mM in DMSO

Preparation of the 100 X plate
A 100-fold concentrated dilutions series was prepared in 96-well plate (test item, positive control, negative control).

Preparation of the 4 X dilution plate
The 100 X DMSO plate was diluted 25 fold in a new plate (4 X). For the test item, the level of the DMSO is adjusted to 1% final.

Contact between the cells and the test and reference items: replacement of 150 µl treatment medium for the 5 seeded plates.
4 X plate: replicated 5 times. Volume: 50 µl
Plate 1X: covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37°C, 5% CO2).

DAY 4: LUCIFERASE ACTIVITY
After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then 100 µl of luciferase substrate (luciferine + ATP + lysing agent) were then added in each well.
New incubation time: 15 min at room temperature.
Measurement with a luminometer.

CELL VIABILITY ASSESSMENT (MTT DYE TEST):
MTT solution: diluted at 0.6 mg/ml in treatment medium
MTT volume: 225 µl
MTT incubation: 4 hours (+/-30 min) at 37°C, 5% CO2
Elimination of the staining solution and treatment with 200 µl of 10% SDS one night in the dark (37°C, 5% CO2)
Measurement of the absorbance at 540 nm.

ACCEPTABILITY CRITERIA

- Positive control:
o Imax > 1.5 in at least one dose,
o EC1.5 within two standard deviations of the historical mean : 2.9 µM ≤EC1.5≤22.6 µM and the mean induction in each run for cinnamaldehyde at 64 µM between 2 and 8.
- Control solvent:
o the average coefficient of variation of the luminescence (CV%) reading for the negative (solvent) control DMSO should be < 20% in each repetition

PREDICTION MODEL

The test item is a potential skin sensitizer if the 4 following parameters are met in 2/2 or 2/3 repetitions. Otherwise the prediction is concluded negative:
1. Imax> 1.5 and statistically significantly different as compared to the solvent control (as determined by a 2-tailed, unpaired Student’s T-test)
2. Cellular viability > 70% for Imax> 1.5 (lowest concentration)
3. EC1.5 < 200 µg/ml
4. Apparent overall dose-reponse for luciferase induction
Positive control results:
Positive control: mean EC1.5 13.19 (geometric mean of 14.31, 11.24, 14.26) and mean Imax 2.99 (3.40, 2.95, 2.61)
Positive controls are valid and in acceptance criteria
Run / experiment:
other: 1
Parameter:
other: Imax
Value:
1.03
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 2
Parameter:
other: Imax
Value:
1.06
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 3
Parameter:
other: Imax
Value:
0.97
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 1
Parameter:
other: Viability (IC70 µg/ml)
Value:
305.14
Vehicle controls validity:
valid
Run / experiment:
other: 2
Parameter:
other: Viability (IC70 µg/ml)
Remarks on result:
not determinable because of methodological limitations
Run / experiment:
other: 3
Parameter:
other: Viability (IC70 µg/ml)
Value:
353.92
Vehicle controls validity:
valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Recommended substances for demonstrating technical proficiency with the Keratinosens™ test method were tested with success.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: CV% < 20% for each repetition
- Acceptance criteria met for positive control: mean EC1.5 = 13.19 within two standard deviations of the historical mean : 2.9 µM ≤EC1.5≤22.6 µM and the mean induction in each run for cinnamaldehyde at 64 µM between 2 and 8 (3.40, 2.95 and 2.61)
- Range of historical values if different from the ones specified in the test guideline: between 2.9 µM ≤EC1.5≤22.6 µM

IC70 in repetition 2 was very different from the one of other repetition.

IC70 was calculated plate by plate and it clearly appeared that the plate 2 in repetition 2 showed a higher toxicity than the other plates. Therefore a 3rd repetition was conducted.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the retained experimental conditions, 3/3 repetitions were negative and the test item X330 was consider as not skin sensitizer. The test method Keratinosens ™ is considered scientifically valid to be used as part of an integrated approach to support the identification of the sensitization potential of test item for hazard classification and labeling purposes.
Executive summary:

The test item X330 was tested for its potential to induce skin sensitization in a strategy of assay. It was tested according to OECD guideline 442D, in vitro skin sensitization, ARE-Nrf2 Luciferase Test Method. It addresses the second key event of the skin sensitization AOP.

The test item X330 dissolved in saline solution was tested at 12 concentrations, from 0.2 µg/ml to 400 µg/ml.

the study decomposed in 3 independant repetitions. For each repetition the test item and the reference were replicated on 3 independent plates for the measurement of induction and 2 plates for cytotoxicity purpose.

The 3 repetitions were negative, under the retained experimental conditions, the test item X330 is not classified as not skin sensitizer.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2015
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
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: 040802#
- Expiration date of the lot/batch: 06.08.2016
Purity: 99.17%
- Purity test date: 08/01/2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable in water, hygroscopic
- Solubility and stability of the test substance in the solvent/vehicle: Different vehicles were tested to solubilize the test item but remained less to non-soluble compared to solubility in water. Therefore aqua ad inject containing 2% CMC was used as vehicle.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Yes, incompatibilities exist between the substance and the CMC

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dilution into the vehicule
- Final dilution of a dissolved solid, stock liquid or gel: 12.5, 25 and 50%

FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories GmbH, Venray, The netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: controlled full-barrier maintained breeding system (SPF)
- Age at study initiation:8-9 weeks
- Weight at study initiation:
- Housing: 5 mice/IVC cages
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period:at least 5 days
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3°C
- Humidity (%):55 +/- 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, 12 hours light/ 12 hours dark
Vehicle:
other: water with 2% carboxymethylcellulose (CMC)
Remarks:
AOO, DMSO, PG were tested but found insoluble.
Concentration:
12.5% - 25% - 50%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: a solubility test is performed to define the vehicle and the maximum concentration which is technically applicable to the animals
- Irritation: Tested on 2 animals by topical application on 3 consecutive days, by measurement of the thickness of both ears (without assessment of lymph node proliferation).
+1 animal treated with the vehicle and served as negative control.


- Systemic toxicity: Body weights recorded + daily observation for any clinical signs
- Ear thickness measurements: performed on day 1 (pre-dose), day 3 (48 hours after the 1st dose) and day 6.
Criteria used to consider irritation: Excessive local irritation is indicated by an erythema score ≥ to 3 and/or swelling of ≥ 25%.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- animal assignment: The animals were randomly selected using the validated departmental computerised system E WorkBook (version 9.4.0, ID Business Solutions Ltd.)+ identification (tail)

- Criteria used to consider a positive response: a substance is regarded as a “sensitizer” in the LLNA ifat least 1 concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine – incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (stimulation index equal or greater than 3)

TREATMENT PREPARATION AND ADMINISTRATION:
- Topical application of 25 µl to the entire dorsal surface of each ear.
- Application once daily over 3 consecutive days (first treatment day = day 1).
- Administration of 3H-Methyhl thymidine : on day 5, 20µCi by IV
- Sacrifice: by cervical dislocation, approx.. 5 hours after 3H-methyl thymidine injection.
- Excision of the draining auricular lymph node and collection in PBS.
- Multiple washing procedures.
- Suspension in 1 ml 5% TCA at 4°C for 18 hours.
- Re-suspension in 1 ml 5% TCA and 7 ml scintillation fluid after 1 washing and then storage at room temperature overnight.
- Determination of incorporated 3H-methyl thymidine with a Betacounter and expressed as the number of desintegrations per minute (DPM)
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The stimulation index of the positive control was 9.1. Test is considered valid
Parameter:
SI
Remarks on result:
not determinable

All animals survived throughout the test period without showing any clinical signs.

Incompatibility exists between CMC and chemical containing aluminium. The test was invalidated.

Interpretation of results:
study cannot be used for classification
Conclusions:
In the prescreen test vehicles acetone/olive oil, polyethylene glycol and dimethylsulfoxyde were tested but the test item was found to be less to non-soluble compared to solubility in water. Threrefore, aqua ad inject containing 2% CMC was applied as vehicle. CMC was added in order to reduce surface tension of water, which is required to cover the dermis of the ear with the test item formulation. Incompatibilities of the test item with CMC was detected afterwards. Therefore the results obtained in this study are considered invalid. EC3 value and classification cannot be obtained.
Executive summary:

X330 was tested according to OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay) for skin sensitisation. A prescreen test was performed in order to define the vehicle which will allow to test a minimum of three concentrations of the test substance and in order to keep the test substance in contact with the mouse ears. Following the guideline, acetone/olive oil, polyethylene glycol and dimethylsulfoxyde were tested but the test item was found to be less to non-soluble compared to solubility in water. Threrefore, aqua ad inject containing 2% CMC was applied as vehicle. CMC was added in order to reduce surface tension of water, which is required to cover the dermis of the ear with the test item formulation. Incompatibilities of the test item with CMC was detected afterwards. Therefore the results obtained in this study are considered invalid. EC3 value and classification cannot be obtained. This study cannot be used for classification.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2001
Qualifier:
according to guideline
Guideline:
other: ESAC Statement on the Reduced Local Lymph Node Assay of 27th april 2007.
GLP compliance:
no
Remarks:
Not GLP study because this study was conducted for R&D purposes
Type of study:
reduced LLNA
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:Code X4412
- Expiration date of the lot/batch:n.p.
- test item description: clear colorless liquid

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no treatment
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River, italia
- Females (if applicable) nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known:housed in Standard Pathogen Free (SPF) conditions, in a microbiologically controled animal facility.
- Age at study initiation:8 weeks
- Weight at study initiation:yes
- Housing: Barrier, 4 mice/ in filter top cage
- Diet (e.g. ad libitum):standard laboratory diet
- Water (e.g. ad libitum):filtered drinking water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20
- Air changes (per hr):individually ventilated cages and static microisolator cages
- Photoperiod (hrs dark / hrs light): 12h continuous artificial light
Vehicle:
unchanged (no vehicle)
Concentration:
100%
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: test item is a solution sufficiently persisting after application on mouse ear. It was applied pure
- Irritation: not irritating
- Systemic toxicity: not expected to be directly toxic

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Reduced Local lymph Node Assay (1 concentration tested)
mices divided in 3 groups of 4 animals: 1 test item group, 1 negative control group and one positive control group.
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) .
Results for each treatment groups expressed as the mean Stimulation Index (SI)
SI = ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for negative control group animals.
- Criteria used to consider a positive response: (SI) equal to or greater than 3.0

TREATMENT PREPARATION AND ADMINISTRATION:
No treatment preparation required.
Test item and controls were applied daily for 3 days with a micropipette (25 µl/ear pinnae)

6 days after study initiation, all mice received intravenous injection of 3H-labeled thymidine.
5 hours after: sacrifice and the draining auricular lymph nodes were excised, individually pooled for each animal.
A single cell suspension of lymph nodes is prepared by gentle mechanical disaggregation and cells washed and resuspended in trichloroacetic acid for at least 12h at 4°C.
Precipitates are resuspended in trichloroacetic acid and transferred to an apprropriate scintillation fluid. The incorporation by draining lymph nodes of 3H-labeled thymidine is measured by scintillation counting and recorded as mean disintegrations per minute.
Positive control substance(s):
other: 1-fluoro-2,4-dinitrobenzène (CAS No 70-34-8)
Statistics:
Mean value are used in order to estimate mean(SI) of each group.
Positive control results:
mean (SI) of 0.02% DFNB (positive control) > 3
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Negative control group
Key result
Parameter:
SI
Value:
3.07
Test group / Remarks:
Positive control group
Key result
Parameter:
SI
Value:
1.08
Test group / Remarks:
Test item group
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
Results for each treatment groups expressed as the mean Stimulation Index (SI)
SI = ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for negative control group animals.

BODY WEIGHTS
For negative control group: mean weight (T0)(g) = 14.7 +/- 0.4 - mean weight (T5 days)(g) = 15.7 +/-0.2
For positive control group: mean weight (T0)(g) = 14.2 +/- 0.1 - mean weight (T5 days)(g) = 15.0 +/-0.3
For test item group: mean weight (T0)(g) = 14.7 +/- 0.3 - mean weight (T5 days)(g) = 15.5 +/-0.4
Interpretation of results:
GHS criteria not met
Conclusions:
The mean stimulation index of the 4 animals of the test item group (1.08) was beside 3.
The positive control have a stimulation index above 3.
Consequently, the test item X330 is considered not to be a skin sensitizer under these experimental conditions.
Executive summary:

X330 diluted at 0.4 mol/l with addition of 1.2 mol/l of NaCl was evaluated for the potential to elicit allergic contact dermatitis with the reduced local lymph node assay, following to OECD guideline 429. The test item was applied unchanged (no vehicle).

Species/strain: mice/ CBA:J

Number of animals: 12 (4 in each group)

The mean stimulation index of the 4 animals of the test item group was beside 3.

Consequently, the test item X330 is considered not to be a skin sensitizer under these experimental conditions.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: ISO 10993-10, Biological evaluation of medical devices, Part10: tests for irritation and skin sensitization
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
Buehler test
Justification for non-LLNA method:
Used according to 93/42/EC directive because of the mode of application which is closer to the one of the medical device tested and because of problem of solubility in the different vehicle of the LLNA (see skin sensitization.003 record)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:Code X113 batch D970516C
- Expiration date of the lot/batch: 30 May 2019
- test item description: Amphoteric salt solution with proprietary makeup which contains 0.1 mol/l of X330 and 0.3 mol/l NaCl
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
Room temperature
Stable under test conditions
Physical description: liquid
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no treatment
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elm Hill Labs
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: young adult
- Weight at study initiation: between 301g and 395 g
- Housing: per group
- Diet (e.g. ad libitum): daily
- Water (e.g. ad libitum): ad libitum
- Acclimation period: > 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 26
- Humidity: 30-70%
- Photoperiod (hrs dark / hrs light): 12h continuous artificial light
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.3 ml
Day(s)/duration:
1st day/ 6 hours (+/- 30 min)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.3ml
Day(s)/duration:
3rd day/6 hours (+/- 30 min)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.3ml
Day(s)/duration:
5th day/6 hours (+/- 30 min)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.3ml
Day(s)/duration:
8th day/6 hours (+/- 30 min)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.3ml
Day(s)/duration:
10th day/6 hours (+/- 30 min)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.3ml
Day(s)/duration:
12th day/6 hours (+/- 30 min)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.3ml
Day(s)/duration:
15th day/6 hours (+/- 30 min)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.3ml
Day(s)/duration:
17th day/6 hours (+/- 30 min)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.3ml
Day(s)/duration:
19th day/6 hours (+/- 30 min)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.3ml
Day(s)/duration:
33th day/ 6 hours (+/- 30 min)
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 9
- Test groups: 10 animals
- Control group: 5 animals
- Site: left flank of 10 animals
- quantity of test item: 0.3 ml (no vehicle) via Hill Top Chamber
- Frequency of applications: 3 times a week during 3 consecutive weeks
- Duration: 6 hours of application
- Concentrations: unchanged

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 14 days after the final induction
- Test groups: 10 animals
- Control group: 5 animals
- Site: ventral flank of each test and control animal
- quantity of test item: 0.3 ml (no vehicle) via Hill Top Chamber
- Site: dorsal flank of each test and control animal
- quantity of control item: 0.3 ml
- Evaluation (hr after challenge): 24
Challenge controls:
A non-woven cotton disk inside a Hill Top Chamber® was saturated with 0.3 mL of the test article and topically secured with hypoallergenic tape to the intact skin on the ventral flank of each test and control animal. The control article was similarly patched to the dorsal flank of each test and control animal. The trunk of each animal was wrapped with an elastic band to hold the occluded patches in place.
Positive control substance(s):
yes
Remarks:
periodic positive control study is performed at the same lab facility and under same study protocol.
Positive control results:
A positive control study for the closed patch sensitization test was performed at the same lab facility and under same study protocol from 12 July 2017 to 11 August 2017. DNCB is used as positive control. All of the ten test animals demonstrated a positive sensitization response to the known sensitizer, DNCB.
None of the control animals demonstrated a sensitization response.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
test item site, unchanged (no vehicle)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no findings
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
test item site, unchanged (no vehicle)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no findings
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
not concerned (negative control)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no findings
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
not concerned (negative control)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no findings
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
control site (0.9% NaCl)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no findings
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
control site (0.9% NaCl)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no findings
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
The test item showed no evidence of causing delayed dermal contact sensitization in the guinea pig.
Executive summary:

X330 diluted at 0.1 mol/l was evaluated for the potential to elicit delayed dermal contact sensitization in the guinea pig. This study was conducted based on the requirements of ISO 10993-10, Biological evaluation of medical devices, Part 10: Tests for irritation and skin sensitization.

The test item was occlusively patched to the intact skin often animals for 6 hours (±30 minutes), three times a week, over a 3 week period. The control article (NaCl 0.9%) was similarly patched to five animals. Following a 2-week recovery period, the ten test and five control animals were occlusively patched with the test article and the control article. All sites were observed for evidence of dermal reactions at 24 and 48 hours after patch removal.

The test article showed no evidence of causing delayed dermal contact sensitization in the guinea pig.

Endpoint:
skin sensitisation: in chemico
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Endpoint:
skin sensitisation, other
Remarks:
Clinical evaluation of a medical device
Type of information:
not specified
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
other: MEDDEV 2.7.1- Clinical evaluation: a guide for manufacturers and notified bodies under directives 93/42/EEC and 90/385/EEC
Version / remarks:
revision 4, June 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other:
Principles of method if other than guideline:
Pursuant to
- section 6a of Annex I to Directive 93/42/EEC (amended by Directive 2007/47/EC) and to
- section 5a of Annex 1 to Directive 90/385/EEC (amended by Directive 2007/47/EC),
the demonstration of conformity with Essential Requirements for a medical device must include a clinical evaluation, which is conducted in accordance with Annex X to Directive 93/42/EEC.
The MEDDEV 2.7.1 promotes a common approach to clinical evaluation for medical devices regulated by directives 90/385/EEC and 93/42/EEC.
The depth and extent of clinical evaluations should be flexible and appropriate to the nature, intended purpose, and risks of the device in question. Therefore, this guidance is not intended to impose device-specific requirements.
Clinical evaluation is a methodologically sound ongoing procedure to collect, appraise and analyse clinical data pertaining to a medical device and to analyse whether there is sufficient clinical evidence to confirm compliance with relevant essential requirements for safety and performance when using the device according to the manufacturer’s instructions for use.
Justification for non-LLNA method:
human data are available, especiallyfrom a clinical study on 150 volunteers following the Marzulli-Maibach Human Repeat Insult Patch Test
Species:
other: Human
Strain:
other: not concerned
Sex:
male/female
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
Pure X113 (40.4 g/mol of X330)
No. with + reactions:
22
Total no. in group:
161
Clinical observations:
Nb with erythema edema (quotation 1): 10, Nb with erythema, edema and vesicles (quotation 2): , Nb with strong reaction (bullae) (quotation 3+): 8
Remarks on result:
no indication of skin sensitisation

Regarding the clinical study performed on 164 volunteers following the Marzulli-Maibach Human Repeat Insult Patch Test, with 8 occlusive applications with duration of 48 h of application, during 3 weeks of induction phase and a challenge test 2 weeks after. This study concludes that the batch tested of X113 did not induce clinically significant skin irritation nor show any evidence of induced allergic contact dermatitis in human subjects.

Into all the clinical data assessed of the medical device from materiovigilance system, no evidence of sensitisation was reported.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification

Discussion

Over the last decades, predictability tests for sensitizing nature of chemical substances have gone from a classic human clinical evaluation to well-defined animal tests. LLNA test is considered a "gold standard". Innovative, diversified and more mechanistic in vitro tests are being developed, in particular using the expression of intracellular biological processes in connection with gene marker activation and identification of newly updated pre-teonomic markers. It is appropriate to use these tests in multiple combinations to compare and strengthen the results.

The current knowledge of the chemical and biological mechanisms associated with skin sensitization has been summarized as an AOP. From this AOP, the first key event cannot be explored as DPRA test is not recommended with metal and complex substances. Nevertheless, the molecular structure of X330 shows that X330 is unlikely to act as an electrophilic substance. The second key event, activation of the Keap1-Nrf2-ARE pathway, the Keratinosens® test, is negative for the test substance X330. Nevertheless, X330 is also found to activate THP-1 cells under test conditions of the human Cell Line Activation Test (h-CLAT), corresponding to the third key event. Information alone from these both tests is not sufficient to conclude on the presence or absence of skin sensitization potential of chemicals. But results of these both tests are in opposition. Therefore no conclusion can be drawn from these integrated approaches.

Moreover, the LLNA assay present limit when the test item X330 needs to be solubilized. One assay, non-GLP realized on a single concentration showed negative results with a diluted solution (X4412 formulation).

2 in vivo tests performed on guinea pigs following European guidelines conclude on the non- sensitizing properties of X330 in its X113 formulation.

Clinical data, available from the clinical assessment of X113 used since more than 20 years as medical device confirms the safety of the mixture. Especially, a clinical study performed on 55 volunteers following the Marzulli-Maibach Human Repeat Insult Patch Test, with 8 to 9 weeks of induction phase and a challenge test 2 weeks after. This study concludes that the batch tested of X113 did not induce clinically significant skin irritation nor show any evidence of induced allergic contact dermatitis in human subjects.

With this weight of evidence we can conclude on the molecule X330 safety in terms of skin sensitization.