Aluminate(2),[[N,N'-1,2-ethanediylbis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON]hydroxy-,disodium

Endpoint summary

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 (Klimisch 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	1
Skin sensitization.001	h-CLAT	2018	Y	Y	Liquid (from 848 to 3040 µg/mL in saline solution)	Y	POSITIVE	Activation of the dendritic cell with both CD-86 and CD-54 markers on 2 independent runs. (exclusion of the 3040 µg/ml value because of cytotoxicity)	1
Skin sensitization.002	KeratinoSens	2018	Y	Y	Liquid (from 0.2 to 400 µg/ml in saline solution)	Y	NEGATIVE	2/3 repetitions negative	1
Skin sensitization.005	ISO Closed Patch Sensitization Study in Guinea Pig	2017	Y	Y	Liquid (56.7 g/l in saline solution) (The tested product is X1.504.5, which is diluted X300 in saline solution)	Y	NEGATIVE	-	1
Skin sensitization.004	ISO Closed Patch Sensitization Study in Guinea Pig	2014	Y	Y	Gel (113.4 g/L in saline solution) (The tested product is W330, which is diluted X300 in saline solution stabilized with glycine and jellified with 0.6% carrageenan)	Y	NEGATIVE	-	1

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-2018

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:

Yes. 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

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: #070601
- Expiration date of the lot/batch: 06/06/2019
- Purity test date: 92.7%

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 and 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,: 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL for the cytotoxicity test (XTT test) and 848, 1018, 1222, 1466, 1759, 2111, 2533 and 3040 for the main experiment

FORM AS APPLIED IN THE TEST: liquid (powder dissolved in saline)

Details on the study design:

TEST SYSTEM: THP-1 cells,
SOURCE SPECIES: human
CELL TYPE: Leukemic monocyte
SOURCE: ATCC, #TIB-202
JUSTIFICATION OF THE TEST SYSTEM USED:
THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.

VEHICLE:
Saline solution (0.9% NaCl).
JUSTIFICATION OF THE CHOICE OF THE VEHICLE: test item is soluble in saline solution and insoluble in DMSO.
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)

CONTROLS
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

PREPARATION OF THE CELLS
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.

CYTOTOXICITY ASSAY
Dose finding assay: XTT test
Number of test: 2
Volume of test item and each controls applied: 100 µl
Number of replicates: 7
Incubation time: 24 hours

XTT concentration: XTT buffer solution and substrate solution mixed right before application at a ratio 1:100
Volume added: 50µl
Spectrophotometer:
Wavelenght: 450 nm (reference wavelength 690 nm)
Determination of the absorbance value: SoftMax Pro Enterprise (version 4.7.1)

ACCEPTABILITY OF THE CITOTOXICITY ASSAY:
• mean absorbance of the medium control is ≥ 0.5
• mean viability of the solvent control is ≥ 90% in comparison to the medium control

MAIN TEST:
Number of replicate :2
Dose of the test item: 1.2 × CV75
Range of concentrations: 7 dilutions were prepared by serial 1:1.2 dilution in saline. All concentrations were further diluted 1:50 in culture medium.
Concentrations applied:
Volume of each test item : 500 µl
Volume of cells: 500 µl
Time of incubation: 24 hours
Staining of the cells: with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
Flow cytometry acquisition: The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

ACCEPTANCE CRITERIA FOR THE MAIN TEST
The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
• For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.

PREDICTION MODEL
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE

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 2 runs (µg/ml)

Parameter:

other: mean CV75 value

Remarks:

XTT tests

Value:

2 533.35

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Key result

Run / experiment:

other: 1

Parameter:

other: RFI(%) CD54

Remarks:

for all tested concentrations

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: RFI (%) CD54

Remarks:

not for all tested concentrations

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 1

Parameter:

other: RFI (%) CD86

Remarks:

for all tested concentrations

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 (%) CD86

Remarks:

for all tested concentrations

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

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%

	run 1			run2
conc. (µg/ml)	RFI (%) CD54	RFI (%) CD86	cell viability (%)	RFI (%) CD54	RFI (%) CD86	cell viability (%)
848	234.8	224.1	90.4	173.0	186.1	90.5
1018	247.0	274.6	87.0	186.5	216.4	89.1
1222	283.3	323.7	81.6	209.0	229.9	89.9
1466	287.9	268.9	80.3	242.7	361.3	85.9
1759	253.0	304.8	66.2	205.6	319.7	82.0
2111	307.6	324.6	74.0	240.4	441.6	73.5
2533	322.7	379.8	62.2	277.5	491.6	64.0
3040	359.1*	545.2 *	42.2	321.3*	773.7*	47.5

*: as cell vaibility below 50%, these results are excluded from the evaluation

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item X300 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 X300 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 X300 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 X300 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 2533.35 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):

848, 1018, 1222, 1466, 1759, 2111, 2533 and 3040 µg/ml.

The test item X300 was tested in 2 independent runs. The highest tested test item concentration (3040 μg/mL) of both h-CLAT runs 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. The RFI of CD54 was greater than 200% in all concentrations of the first run and in the most concentrations of the second run. 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 X300 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).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-2018

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)

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: #070601
- Expiration date of the lot/batch: 06/06/2019
- Purity test date: 92.7%

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 and stable in water or saline

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)

Gene induction > 1.5
EC1.5 into the lab historical data

Key result

Run / experiment:

other: 1

Parameter:

other: IC70 (µg/ml)

Value:

284.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: IC70 (µg/ml)

Value:

228.41

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 3

Parameter:

other: IC70 (µg/ml)

Value:

379.08

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

1.02

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

1.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 3

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

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

As response was different between the 2 first repetitions, a third repetition was conducted in order to conclude.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the retained experimental conditions, 2/3 repetitions were negative and the test item X300 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 X300 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 X300 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.

2/3 repetitions were negative, under the retained experimental conditions, the test item X300 is not classified as not skin sensitizer.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-2017

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:

no

GLP compliance:

yes

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: 040801#
- Expiration date of the lot/batch: 05.08.2016
Purity: 91.61%
- 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 (see prescreen test).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Yes, incompatibilities exist between the substance and the CMC (see conclusion of the present record)

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: 2% CMC in aqua ad injected

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 aluminum. 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. Therefore, 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 were detected afterwards. Therefore the results obtained in this study are considered invalid. EC3 value and classification cannot be obtained.

Executive summary:

X300 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. Therefore, 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 were 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.

Reference 4

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)

Deviations:

no

GLP compliance:

yes

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 X1.504.5 batch F970403C
- Expiration date of the lot/batch: 02/5/2019
- test item description: Amphoteric salt solution with proprietary makeup which contains 0.15 mol/l of X300 and 0.45 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 between 314g and 486 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:

100% / 0.3ml

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:

100% / 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:

100% / 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:

100% / 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:

100% / 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:

100% / 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:

100% / 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:

100% / 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:

100% / 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:

100% / 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. 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

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

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

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

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:

X300 diluted at 0.15 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.

Reference 5

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013-2014

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

Version / remarks:

01/09/2002

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

Used according to 93/42/EC directive because of the registration of the test item, mode of application which is closer to the one of the medical device tested.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Code GDO batch 030702
- Expiration date of the lot/batch: January 2014
- test item description: semi-solid gel which contains 0.3 mol/l of X300 and 0.3 mol/l of a conservative (glycine) and 0.6% of carrageenan
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
Room temperature and protected from light
Stable under test conditions
Physical description: semi-solid gel
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material during the preliminary study: diluted at 25, 50, 75 and 100% in 0.9% sodium chloride solution.
- Treatment of test material prior to testing (mean experiment): no treatment

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France
- Males
- Age at study initiation: young adult
- Weight at study initiation: between 323g and 388 g
- Housing: per group
- Diet (e.g. ad libitum): daily
- Water (e.g. ad libitum): ad libitum
- Acclimation period: > 5 days
- Number of animals per group:
- Preliminary study: 2 males
- Main study treated group: 10 males
- Negative control group: 5 males
- Positive control group (at least every 6 months): 5 males
Preparation of animals: The day prior to each treatment, an area of approximately 24 cm2 (4 cm x 6 cm) on both flanks (induction and challenge areas) were clipped free of hair using an electric clipper. Only healthy animals with an intact skin were used for the experiment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity: 45-65%
- Photoperiod (hrs dark / hrs light): 12h continuous artificial light (except one night because of the switch to winter time)

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100% / 0.5ml

Day(s)/duration:

1st day/6 hours

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:

100% / 0.5 ml

Day(s)/duration:

2nd day/ 6 hours

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:

100% / 0.5 ml

Day(s)/duration:

3rd day/6 hours

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:

100% / 0.5 ml

Day(s)/duration:

8th day/ 6 hours

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:

100% / 0.5 ml

Day(s)/duration:

9th day/6 hours

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:

100% / 0.5 ml

Day(s)/duration:

10th day/6 hours

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:

100% / 0.5 ml

Day(s)/duration:

15th day/6 hours

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:

100% / 0.5 ml

Day(s)/duration:

16th day/6 hours

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:

100% / 0.5 ml

Day(s)/duration:

17th day/6 hours

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:

100% / 0.5 ml

Day(s)/duration:

31th day/ 6 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10

Details on study design:

PRELIMINARY STUDY
- No. of animals: 2
- Tested concentrations: 25%, 50%, 75% in 0.9% NaCl and 100%
- Occlusive patch during 6 hours
- Grading of the application sites for erythema and oedema using the Magnusson and Kligman grading scale at 24h and 48h after patch removal.

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: 0.5 ml (no vehicle) with a piece of absorbent gauze, held in place with an occlusive dressing (BLENDERM 3M 5 cm width)+adhesive tape Elastoplate®.
- 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: 13 days after the final induction
- Test groups: 10 animals
- Control group: 5 animals
- Site: right flank region
- quantity of test item: 0.5 ml (no vehicle) with a piece of absorbent gauze, held in place with an occlusive dressing (BLENDERM 3M 5 cm width)+adhesive tape Elastoplate®.
- quantity of control item: 0.5 ml
- Evaluation (hr after challenge): 24 and 48

Challenge controls:

Under the same conditions as above, negative control animals received the test item at 100% and positive control animals received 0.5 mL of 1% DNCB (2,4-dinitrobenzene solution.

Positive control substance(s):

yes

Remarks:

1% DNCB (2,4-dinitrobenzene) solution

Positive control results:

In the positive control group, at time 24 hours, 1 out of 5 animals showed discrete or patchy erythema (score 1), 3 out of 5 animals showed moderate erythema (score 2) and 1 out of 5 animals showed intense erythema (score 3). At time 48 hours, 5 out of 5 animals showed moderate erythema (score 2). So 100% of animals became sensitized with DNCB. The study is valid.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no specific findings

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no specific findings

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no specific findings

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no specific findings

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

1%

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

no specific findings

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

1%

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

no specific findings

Remarks on result:

positive indication of skin sensitisation

PRELIMINARY STUDY:

The concentration of 25% (v/v), 50% (v/v), 75% (v/v) and 100% (undiluted) were tested. No skin reaction was seen whatever the concentration tested.

In conclusion, the highest concentration that caused no more than slight erythema when tested by cutaneous application was 100% (undiluted). It was therefore the concentration chosen for the induction phases.

The highest concentration that produced no erythema by cutaneous application is 100%. It is therefore the concentration chosen for the challenge phase on D31.

MAIN STUDY:

There was no skin change at times 24 and 48 hours in animals of the negative control group or in animals treated during the challenge phase with the test item at 100%.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions adopted and according to ISO 10993-10 (Buehler test), the test item was found to be non sensitising in the Guinea pig.

Executive summary:

X300 diluted at 0.3 mol/l into a semi-liquid gel form 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 experimental technique is based on intact (unabraded) skin and depends on measurement of the extent and degree of skin reaction to a challenge exposure following previous topical exposure in the absence of any adjuvant.

The test item was occlusively patched to the intact skin of ten animals for 6 hours, 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 as challenge test. All sites were observed for evidence of dermal reactions at 24 and 48 hours after patch removal.

Under these experimental conditions, the test item was found to be non sensitizing in the Guinea pig.

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 X300 shows that X300 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 X300. Nevertheless, X300 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. And 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 X300 needs to be solubilized in order to stay on the mouse’s ear.

2 in vivo tests performed on guinea pigs following European guidelines conclude on the non-sensitizing properties of X300 in its X1.504.5 formulation and W330 formulation.

With this weight of evidence we can conclude on the molecule X300 safety in terms of skin sensitization.