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Skin sensitisation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Cytidine 3'-(dihydrogen phosphate)
EC Number:
200-556-6
EC Name:
Cytidine 3'-(dihydrogen phosphate)
Cas Number:
63-37-6
Molecular formula:
C9H14N3O8P
IUPAC Name:
cytidine 3'-(dihydrogen phosphate)
Test material form:
solid
Details on test material:
Synonyms: 5-CMP acid, 5'-Cytidylic acid, Cytidine 5'-monophosphate
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

In vitro test system

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.

Results and discussion

Positive control results:
Imax = 3.59
IC1.5 = 20.47 (geometric mean)

In vitro / in chemico

Resultsopen allclose all
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.

Any other information on results incl. tables

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.

 

Applicant's summary and conclusion

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.

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