Registration Dossier

Administrative data

Description of key information

In chemico skin sensitisation (OECD 442C):

Data waiving. Study technically not feasible: The test can not be conducted due to solubility problems in the test conditions.

In vitro skin sensitisation:

Weight of evidence: OECD 442D. GLP study. The test item showed a Imax of 1.37 and a IC50 and IC30 higher than 2000 µM in KeratinoSens™. Therefore, under this conditions the test item may be classified as not skin sensitizer.

Weight of evidence: OECD 442E. GLP study. Under the experimental conditions of this study, the test item was found to be negative in the h-CLAT up to 3000 µg/mL. Therefore, the test item may be classified as not skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
Study technically not feasible: The Direct Peptide Reactivity Assay (DPRA) (OECD TG 442C) can not be conducted due to solubility problems in the test conditions. According to Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a – Version 6.0, this test is only applicable to substances that are soluble in an appropriate solvent at a final concentration of 100 mM. A solubility test was conducted with various solvents; acetronile, water, water/acetronile 1:1, isopropanol, acetone, acetone/acetronile 1:1, DMSO and acetronile, with and without sonication. The test item was very difficult to solubilize and there was precipitate after 1 hour in all conditions.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April 18, 2017 - April 28, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ECVAM DB-ALM protocol 155: KeratinoSens
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes
Details on the study design:
- Cell line used: KeratinoSens™ (Givaudan) cultured in maintenance medium (DMEM 1 g/l glucosa, 9.1% non-heat inactivated foetal calf serum, 0.05% geneticin - stored at 5ºC ± 3°C) at 37ºC, 5% CO2. Cells were exempt of mycoplasma.
- Passage number and level of confluence of cells: cells were used at passage 15 in repetition 1, passage 17 in repetition 2 and passage 19 in repetition 3.
- Cell counting was performed using a Malassez cell (grid of 10 x 10 tiles and a total volume of 1 mm3), cells were counted on two lines (one vertical and one horizontal) and cells suspension was adjunted at 8E04 cells/ml in seeding medium (DMEM 1 g/l glucosa, 9.1% non-heat inactivated doetal calf serum - stored at 5ºC ± 3°C). 10E04 cells/ml were distributed in 3 white cell culture plates (96 wells) for the induction measurement and two transparent cell culture plates (96 wells) to assess the cytotoxicity. In order to ensure a good homogeneity of seeding, cells suspension was regularly mixed all along the seeding. The seeded plates were incubated for 24 hours ± 1 hour at 37°C, 5% CO2.
- Luminometer used: GloMax™ (Promega)
MULTISKAN EX plate reader (Thermo life sciences) - reading range 0 - 3.5 units of Absorbance -Blue formazan (CAS # 57360-69-7) linearity range at 540 nm: 0 - 2.200 units of Absorbance
- Number of repititions and replicates: the study was composed of three independent repetitions. For each repetition the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with fresh stock solution.
- Test chemical concentrations:
The test item was prepared directly at 1295.8 µM and 1000 µM (1X) in treatment medium 1% DMSO. Positive control was diluted 100-fold in DMSO (Sigma Aldrich Batch no. SZBG2170) from a 6.4 mM stock solution and then diluted 25-fold in a new plate in treatment medium and then further diluted 4-fold in the seeding plate.
Test item was tested at 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 1295.8 µM.
Negative control: 6 wells of solvent control,1% DMSO in treatment medium (DMEM 1 g/l glucose, 1% non-heat inactivated foetal calf serum - stored at 5°C ± 3°C), with cells and 1 well of solvent control without cells.
Positive control: 5 concentrations of Cinnamaldehyde (Sigma Aldrich Batch no. MKBT8955V) on each culture plate. The concentration varies from 4 to 64 µM according to a geometric progression of ratio 2.

- Description of evaluation and decision criteria used:
The test item is identified as potential skin sensitizer if the 4 following conditions are met in 2 of 2 or 2 of 3 repetitions. Otherwise the keratinosens™ prediction is cosidered as negative:
1) The Imax is strictly 1.5 fold higher of the basal luciferase activity* statistically significantly to the value obtained for the negative control (as determined by a two-tailed, unpaired Student's t-test on the raw RLU values). If the Imax is exactly equal to 1.5, the test item is rated as negative and no EC1.5 value is calculated.
2) The EC1.5 value is strictly below 1000 µM (or <200 µg/ml for the test item with no defined molecular weight)
3) At the lowest concentration with a gene induction above 1.5, the cell viability must be strictly above 70% (i.e EC1.5 < IC70).
4) There is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.

- Description of study acceptance criteria used:
1) Positive control
- The gene induction must be statistically significant above the threshold of 1.5 in at least one dose.
- The EC1.5 value should be between IDEA Lab historical data: mean EC1.5 value ± 2 SD and the average induction, in each repetition, for cinmamaldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
2) Negative control: for each repetition, the coefficient of variation of the solvent controls (3 x 6 wells) must be less than 20%. If for one repetition the validity criteria is not met, a third repetition should be considered.

Positive control results:
Imax = 3.59
IC1.5 = 20.47 (geometric mean)
Key result
Run / experiment:
mean
Parameter:
other: Imax
Value:
1.37
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Imax lower than 1.5, no EC1.5 is calculated
Key result
Run / experiment:
other: geometric mean
Parameter:
other: IC50
Value:
2 000
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: geometric mean
Parameter:
other: IC30
Value:
2 000
Vehicle controls validity:
not applicable
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: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: recommended substances for demonstrating technical proficiency with the KeratinoSens™ test method were tested to validate the method.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, for each repetition, the coefficient of variation of the solvent controls (3 x 6 wells) was less than 20% in repetition 1 and 3. However, in repetition 2, Imax and EC1.5 fulfilled validity criteria and Control solvent CV was higher than expected value (>20%) (because of higher RLU values on one of the three culture plates). This criteria is to demonstrate the homogeneity of the plates. If CV of each plate was higher than 20% it could lead to false results but CV being <<20% there is no risk since inductions are calculated plate by plate. The low CVs of each plate allow us to validate the repetition. Nevertheless, a third repetition was conducted to confirm results.
- Acceptance criteria met for positive control: yes, OECD guideline data set validation provides a positive control EC1.5 interval from 7 µM to 30 µM, for repetition 1 and 3, EC1.5 are slightly higher than the current laboratory historical data range (1.9 µM ≤ EC1.5 ≤ 23.3 µM) but are however in the 7-30 µM interval which allows us to validate the repetition.
Repetition 1: Imax is slightly lower than the expected value (<2) but we can observe a clear dose-response with increasing luciferase activity inductions which allows to validate the test.
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: Mean Imax = 5.29 ± 3.12; Mean EC1.5 = 12.59 ± 5.36, Mean IC70 > 64 µM; N= 117.

TTable 1. Positive control results.

Cinnamaldehyde

4 µM

8 µM

16 µM

32 µM

64 µM

EC1.5

Imax

Rep 1

1.03

1.11

1.26

1.56

1.94

28.67

1.94

Rep 2

1.31

1.32

1.74

2.29

6.54

11.43

6.54

Rep 3

1.20

1.13

1.29

1.62

2.30

26.16

2.30

Mean

1.18

1.18

1.43

1.82

3.59

20.47*

3.59

* geometric mean

Table 2. Negative control results.

Control solvent

CV %

control solvent

Rep 1

10.89

Rep 2

24.77

Rep 3

16.17

 

Table 3. Coefficient of variation of the negative controls.

Control solvent

CV % control solvent

Plate 1

Plate 2

Plate 3

3 plates

Rep 1

7.7%

9.4%

5.4%

10.89%

Rep 2

6.1%

11.6%

6.2%

24.77%

Rep 3

2.7%

11.9%

10.8%

16.17%

 

Table 4. Test item results.

 

VIABILITY

INDUCTION

 

IC50 µM

IC70 µM

Imax

Linear EC1.5 µM

EC1.5 Lin/Log µM

Rep 1

> 2000

> 2000

1.39

-

-

Rep 2

> 2000

> 2000

1.09

-

-

Rep 3

> 2000

> 2000

1.64

1196.90

1146.23

Mean

-

-

1.37

-

-

Geometric mean

> 2000

> 2000

-

-

-

Repetition 1 and 2 Imax are lower than 1.5, no EC1.5 is calculated.

Repetiton 3, Imax is higher than 1.5, EC1.5 is equal to 1196.90 µM. According to the prediction criteria, EC1.5 being higher than 1000µM the repetition is considered as non sensitizer.

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test item showed a Imax of 1.37 and a IC50 and IC30 higher than 2000 µM in KeratinoSens™. Therefore, under this conditions the test item may be classified as not skin sensitizer.
Executive summary:

The test method KeratinoSens™ has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item according to OECD 442D, following GLP. A skin sensitiser is a substance that leads to an allergic response following skin contact. This allergic response includes inflammatory responses as well as gene expression associated with specific cell signaling pathways in the keratinocytes. One of the target genes is AKR1C2, and the test method KeratinoSens™ is based on the evaluation of the activation of this gene in transformed keratinocytes by monotoring the induction of the luciferase gene fused to AKR1C2. After 48 h of contact between the test item with KeratinoSens™ monolayer, the induction of the luciferase is quantified. A positive control and negative control were run in parallel, as well as a cytotoxicity assay. Three repetitions of the assay was performed on different days. The test item showed a Imax of 1.37 and a IC50 and IC30 higher than 2000 µM in KeratinoSens™. Therefore, under this conditions the test item may be classified as not skin sensitizer.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 27, 2017 - October 24, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD guideline No. 442E: "In vitro skin sensitization: human Cell Line Activation Test (h CLAT)"
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: DB-ALM Protocol No. 158: human Cell Line Activation Test (h-CLAT)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes
Details on the study design:
VEHICLE AND POSITIVE CONTROLS

- Vehicle: Based on the results of the solubility assay, the vehicle selected was saline (0.9% NaCl, Lavoisier).
- Positive controls: 2,4-Dinitrochlorobenzene (DNCB) (Sigma-Aldrich; CAS No. 97-00-7; Purity ≥ 99%) and Nickel Sulfate (NiSO4) (Merck; CAS No. 10101-97-0, Purity ≥ 99%).

TEST ITEM AND CONTROLS PREPARATION

- Positive controls:
1) DNCB was prepared at the concentration of 8 µg/mL in DMSO: on the treatment day,DNCB was mixed with DMSO at a concentration of 2 mg/mL and this solution was then 250-fold diluted in cRPMI in order to obtain a 8 µg/mL stock solution.
2) NiSO4 was prepared at the concentration of 200 µg/mL in 0.9% NaCl: on the treatment day, NiSO4 was mixed with 0.9% NaCl at a concentration of 10 mg/mL and this solution was then 50-fold diluted in cRPMI in order to obtain a 200 µg/mL stock solution.
Both positive control stock solutions were prepared within 4 hours before use, and kept at room temperature and protected from light until use.
- Vehicle control: As 0.9% NaCl was the vehicle selected at completion of the solubility assay, no vehicle control was included in the testing phases and test item formulations data was compared to data obtained from cells treated with culture media (cRPMI).
- Test item: Test item stock formulations prepared in 0.9% NaCl were 100 x concentrated, and then 2 x concentrated formulations were prepared by 1:50 dilution in cRPMI.

CELL LINE AND CELL CULTURE

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

STUDY DESIGN

- Dose-Range Finding assay (DRF): It was performed to assess the test item toxicity and consisted in two separated assays, both were performed as follows:
1) Final concentrations: 23.44, 46.88, 93.75, 187.50, 375, 750, 1500 and 3000 µg/mL.
2) Working solutions: Test item formulations were prepared at 8 different concentrations by 2-fold dilutions using the selected vehicle. These formulations were then diluted 50-fold (as 0.9% NaCl is the selected vehicle) into cRPMI to obtain working solutions.
3) Assay: 500 µL of the working solutions were added to the volume of THP-1 cell suspension in the plate (500 µL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate. The plates were incubated for 24 hours ± 30 minutes at 37ºC and 5% CO2. At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation. The supernatants were discarded and the cells were re-suspended with 600 µL of FACS buffer (D-PBS with 0.1% (w/v) BSA) and the plate was put into the plate-reader of a flow cytometer. A volume of Propidium Iodine (PI) solution at 12.5 µg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 µ/mL per well.

- Main test: Consisted in two validated runs being performed as follows:
1) Final concentrations (both runs): 834.24, 1004.69, 1205.63, 1446.76, 1736.11, 2083.33, 2500 and 3000 µg/mL. The highest concentration corresponded to the highest achievable non-cytotoxic concentration as no CV75 was obtained.
2) Working solutions: Test item formulations were prepared at 8 different concentrations by 1.2-fold dilutions using the selected vehicle. All stock formulations were then 50-fold diluted into cRPMI to obtain working solutions.
3) Positive controls: Working solutions of positive controls CNCB and NiSO4 were prepared.
4) Assay: The exposure was carried out as in the dose-Range Finding assay. At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation, washed twice with 1 mL of FACS buffer and blocked with 600 µL of blocking solution (0.01% globulin in FACS buffer), and incubated at 4ºC for 15 minutes (± 1 minute). After blocking, cells were split in three aliquots of 180 µL into a 96-well round bottom plate and centrifuged before staining with antibodies. A volume of 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IGG1 (isotype) antibodies prepared in FACS buffer was added to each aliquot before incubation for 30 minutes (± 2 minutes) at 4ºC. Finally, cells were washed with 150 µL FACS buffer 2 times and re-suspended in 200 µL FACS buffer. The plate was then positioned into the plate-reader of the flow cytometer. A volume of 10 µL of PI solution at 12.5 µg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 µg/mL per well.
- Flow cytometry analysis.
1) DRF assays: The PI uptake is analyzed using flow cytometry with the acquisition channel B3. A total of 10 000 living cells (PI negative) are acquired. In case of low viability which does not allow obtaining 10 000 living cells, a total of 30 000 events is acquired. Alternatively, cells were acquired for a maximum of 1 minute after the initiation of the acquisition.
2) Main test: The expression of IgG1, CD86 and CD54 was analyzed by flow cytometry with the acquisition channel B1 in order to obtain the Mean Fluorescence Intensity (MFI); whereas the viability (PI uptake) was analyzed with the acquisition channel B3. A total of 10 000 living cells (PI negative) were acquired. When the viability was low and did not allow obtaining 10 000 living cells, a total of 30 000 events was acquired. Alternatively, cells were acquired for a maximum of 1 minute after the initiation of the acquisition. In case cell viability is less than 50%, no MFI is presented in the study report and the corresponding test item concentration are considered too high for interpretation because of the diffuse labelling cytoplasmic structures that are generated following cell membrane destruction.

CALCULATIONS
1) Estimation of the CV75 value: The percentage of living cells (PI negative cells) is used as the value for cell viability.The CV75 value is derived from the dose-response curve as shown in Figure 1 (75% of cell viability, lying between a and c). CV75 is defined as the estimated concentration that is required to elicit 75% cell viability. The CV75 value is calculated by log-linear interpolation utilizing the following equation:
Log CV75= (75 - c) x Log b - (75 - a) x Log d / a -c
2) Main test: Based on the Mean Fluorescence Intensity (MFI), the Relative Fluorescence Intensity (RFI) of CD86 and CD54 were calculated according to the following equation:
RFI = [(MFI of test item-treated (CD86 or CD54) - MFI of test item-treated IgG1) / ((MFI of control-treated (CD86 or CD54) - MFI of control-treated IgG1)] x 100
where:
RFI = Relative Fluorescence Intensity
MFI = Mean Fluorescence Intensity

ACCEPTANCE CRITERIA

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

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

MAIN TEST INTERPRETATION

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

In other circumstances, the run is considered as negative.

PREDICTION MODEL: Based on the individual run conclusions, a final prediction is made as follows:
- if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted,
- if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE (with due consideration of the highest-tested dose conditions) without the need for a third run,
- if however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE.

Positive control results:
- DNCB: The Relative Fluorescence Intensity (RFI) of CD86 and CD54 were 194 and 748 respectively in the run A; and 262 and 379 in the run B. Because RFI(CD86) was higher than 150 and RFI(CD54) higher than 200 in both runs, the result was considered POSITIVE.
- NiSO4: The Relative Fluorescence Intensity (RFI) of CD86 and CD54 were 207 and 1078 respectively in the run A; and 241 and 1426 in the run B. Because RFI(CD86) was higher than 150 and RFI(CD54) higher than 200 in both runs, the result was considered POSITIVE.
Key result
Run / experiment:
other: A
Parameter:
other: RFI (CD86)
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
DNCB = 194, NiSO4 = 207
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: B
Parameter:
other: RFI (CD86)
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
DNCB = 262, NiSO4 = 241
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: A
Parameter:
other: RFI (CD54)
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
DNCB = 748, NiSO4 = 1078
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: B
Parameter:
other: RFI (CD54)
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
DNCB = 379, NiSO4 = 1426
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Precipitation:
1) DRF test: In both tests, at post-treatment observation, no abnormalities such as precipitate/emulsion or cell morphology modification was observed at any tested concentrations.
2) Main test: Run A: no precipitate/emulsion was noted in treated wells. Run B: a yellow coloration of the media was noted in the wells treated with the test item but no morphological abnormality of the cells was noted.

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

Dose-Range Finding results:

Results obtained in the first DRF test (i.e.DRF 1):   flow cytometry measurement after Propidium Iodide staining revealed no cell viability decrease below 75% at any tested concentration. Therefore, no CV75 value was calculated.

 

Results obtained in the second DRF test (i.e.DRF 2):   flow cytometry measurement after Propidium Iodide staining revealed no cell viability decrease below 75% at any tested concentration. Therefore, no CV75 value was calculated.

 

Based on the results from both DRF runs, no mean CV75 was calculated, and the maximum concentration tested in the main test was therefore 3000 µg/mL.

Main test

Table 1. Main test individual results. Run A.

Study No.

Vehicle

Run Letter

Concentration ( µg/mL)

MFI (Geo Mean)

MFI ratio

IgG corrected MFI

RFI (CD86)

RFI (CD54)

Viability (%)

vs. Top control

vs. Top control

IgG

CD86

CD54

CD86/IgG

CD54/IgG

CD86

CD54

vs. cRPMI

vs. DMSO

vs. cRPMI

vs. DMSO

IgG

CD86

CD54

Mean

cRPMI (4)

 

 

 

0.85

4.36

2.05

513

241

3.51

1.20

 

 

 

 

95.4

95.2

95.7

95.4

NiSO4 (2)

0.9% NaCl

 

100

0.93

8.21

13.87

 

 

7.28

12.94

207

 

1078

 

82.8

84.2

83.8

83.6

0.2% DMSO (2)

 

 

 

0.91

5.08

1.55

558

170

4.17

0.64

119

 

53

 

95.8

95.8

96.6

96.0

DNCB

0.2% DMSO

 

4.00

0.88

8.97

5.67

 

 

8.09

4.79

 

194

 

748

73.3

74.5

73.4

73.7

Test item

0.9% NaCl

A

837.24

0.88

4.46

1.71

 

 

3.58

0.83

102

-

69

-

94.5

93.7

94.8

94.4

1004.69

0.90

4.31

1.70

 

 

3.41

0.80

97

-

67

-

93.3

94.4

94.3

94.0

1206.63

0.98

4.31

1.80

 

 

3.33

0.82

95

-

68

-

94.8

93.9

94.8

94.5

1446.76

1.00

4.68

2.33

 

 

3.68

1.33

105

-

111

-

93.4

92.9

93.9

93.4

1736.11

0.98

4.32

1.81

 

 

3.34

0.83

95

-

69

-

94.9

95.2

95.2

95.1

2083.33

0.82

3.95

1.80

 

 

3.13

0.98

89

-

82

-

92.5

92.9

93.0

92.8

2500.00

0.85

3.37

1.74

 

 

2.52

0.89

72

-

74

-

92.0

91.5

91.6

91.7

3000.00

0.84

3.69

2.00

 

 

2.85

1.16

81

-

97

-

93.9

94.0

93.8

93.9

MFI: Mean Fluorescence Intensity

RFI: Relative Fluorescence Intensity

-: not applicable

 

Plate validation criteria:       

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

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

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

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

 

 

 

Table 2. Main test individual results. Run B.

Study No.

Vehicle

Run Letter

Concentration ( µg/mL)

MFI (Geo Mean)

MFI ratio

IgG corrected MFI

RFI (CD86)

RFI (CD54)

Viability (%)

vs. Top control

vs. Top control

IgG

CD86

CD54

CD86/IgG

CD54/IgG

CD86

CD54

vs. cRPMI

vs. DMSO

vs. cRPMI

vs. DMSO

IgG

CD86

CD54

Mean

cRPMI (4)

 

 

 

0.91

5.86

2.02

644

222

4.95

1.11

 

 

 

 

94.4

94.3

94.2

94.3

NiSO4 (2)

0.9% NaCl

 

100

0.85

12.76

16.68

 

 

11.91

15.83

241

 

1426

 

75.8

75.3

75.4

75.5

0.2% DMSO (2)

 

 

 

0.97

5.72

1.94

590

200

4.75

0.97

96

 

87

 

95.6

95.4

95.6

95.5

DNCB

0.2% DMSO

 

4.00

0.88

13.34

4.56

 

 

12.46

3.68

 

262

 

379

77.0

74.6

74.9

75.5

Test item

0.9% NaCl

B

837.24

1.02

5.87

2.23

 

 

4.85

1.21

98

-

109

-

95.0

95.3

95.1

95.1

1004.69

0.94

5.59

2.32

 

 

4.65

1.38

94

-

124

-

95.3

95.2

95.2

95.2

1206.63

0.95

5.28

2.21

 

 

4.33

1.26

87

-

114

-

94.9

94.6

95.1

94.9

1446.76

1.06

5.07

1.95

 

 

4.01

0.89

81

-

80

-

94.6

94.8

95.4

94.9

1736.11

1.00

5.19

1.99

 

 

4.19

0.99

85

-

89

-

94.4

94.4

94.0

94.2

2083.33

1.06

4.75

2.11

 

 

3.69

1.05

75

-

95

-

94.4

93.9

95.3

94.6

2500.00

0.87

4.41

1.93

 

 

3.54

1.06

72

-

95

-

94.2

95.2

95.0

94.8

3000.00

0.89

4.94

2.22

 

 

4.05

1.33

82

-

120

-

93.7

93.7

93.7

93.7

MFI: Mean Fluorescence Intensity

RFI: Relative Fluorescence Intensity

-: not applicable

 

Plate validation criteria:       

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

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

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

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

 

 

 

Table 3. Main test summary results.

Test item Name

Conc. (µ/mL)

RFI for CD86

RFI for CD54

Viability (%)

Run conclusion

General conclusion

A

B

A

B

A

B

A

B

Cytidine 3’-(dihydrogen phosphate)

837.24

102

98

69

109

94.4

95.1

N

N

Negative

1004.69

97

94

67

124

94.0

95.2

1205.63

95

87

68

114

94.5

94.9

1446.76

105

81

111

80

93.4

94.9

1736.11

95

85

69

89

95.1

94.2

2083.33

89

75

82

95

92.8

94.6

2500.00

72

72

74

95

91.7

94.8

3000.00

81

82

97

120

93.9

93.7

N= run with negative outcome

Conc.= concentration

RFI =Relative Fluorescence Index

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item was found to be negative in the h-CLAT up to 3000 µg/mL. Therefore, the test item may be classified as not skin sensitizer.
Executive summary:

An in vitro cell line activation test (h-CLAT) has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item in accordance with the OECD Guideline 442E, following GLP. The h-CLAT method is based on changes in the quantification of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells. A solubility assay with the test item was performed and 0.9% NaCl was chosen as the vehicle. Based on the results from two Dose-Range Finding assays, the upper dose tested was 3000 µg/mL. Two validated successive test runs were performed. In each run, the test item formulations (837.24, 1004.69, 1205.63, 1446.76, 1736.11, 2083.33, 2500 and 3000 µg/mL) were applied to THP-1 cells and cultured for 24 hours and 30 minutes at 37ºC, 5% CO2. Negative and positive controls were run in parallel. After incubation, the expression of CD86 and CD54 was measured by cytometry analysis, and the viability of the cells was determined after being dyed with Propidium Iodine. The Mean Fluorescence Intensity (MFI) was obtained for each test sample and then corrected. The corrected MFI values were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index (RFI) for CD86 and CD54 expression. All validity criteria were met. In both runs, the results of RFI(CD86) and RFI(CD54) were less than 150 and 200 respectively in all concentrations tested, both results were negative. Therefore, the test item was found to be negative in the h-CLAT.

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

In vitro skin sensitisation:

Weight of evidence: The test method KeratinoSens™ has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item according to OECD 442D, following GLP. A skin sensitizer is a substance that leads to an allergic response following skin contact. This allergic response includes inflammatory responses as well as gene expression associated with specific cell signalling pathways in the keratinocytes. One of the target genes is AKR1C2, and the test method KeratinoSens™ is based on the evaluation of the activation of this gene in transformed keratinocytes by monitoring the induction of the luciferase gene fused to AKR1C2. After 48 h of contact between the test item with KeratinoSens™ monolayer, the induction of the luciferase is quantified. A positive control and negative control were run in parallel, as well as a cytotoxicity assay. Three repetitions of the assay was performed on different days. The test item showed a Imax of 1.37 and a IC50 and IC30 higher than 2000 µM in KeratinoSens™. Therefore, under this conditions the test item may be classified as not skin sensitizer.

Weight of evidence: An in vitro cell line activation test (h-CLAT) has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item in accordance with the OECD Guideline 442E, following GLP. The h-CLAT method is based on changes in the quantification of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells. A solubility assay with the test item was performed and 0.9% NaCl was chosen as the vehicle. Based on the results from two Dose-Range Finding assays, the upper dose tested was 3000 µg/mL. Two validated successive test runs were performed. In each run, the test item formulations (837.24, 1004.69, 1205.63, 1446.76, 1736.11, 2083.33, 2500 and 3000 µg/mL) were applied to THP-1 cells and cultured for 24 hours and 30 minutes at 37ºC, 5% CO2. Negative and positive controls were run in parallel. After incubation, the expression of CD86 and CD54 was measured by cytometry analysis, and the viability of the cells was determined after being dyed with Propidium Iodine. The Mean Fluorescence Intensity (MFI) was obtained for each test sample and then corrected. The corrected MFI values were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index (RFI) for CD86 and CD54 expression. All validity criteria were met. In both runs, the results of RFI(CD86) and RFI(CD54) were less than 150 and 200 respectively in all concentrations tested, both results were negative. Therefore, the test item was found to be negative in the h-CLAT and may be classified as not skin sensitizer.

Based on available results, the test item can be considered as not skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available information (negative results in the in vitro studies OECD 442D and OECD442E), the substance is not classified for sensitising properties in accordance with CLP Regulation (EU) No. 1272/2008.