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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:
03 November - 08 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted February 2015
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate
EC Number:
221-309-9
EC Name:
2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate
Cas Number:
3063-94-3
Molecular formula:
C7H6F6O2
IUPAC Name:
2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 449935-248
- Expiration date of the lot/batch: 30 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8ºC)
- Stability under test conditions: stable


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98 µM test substance in DMSO

FORM AS APPLIED IN THE TEST
Solution in DMSO

In vitro test system

Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:

Test System
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Experimental Design
Plating of Cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+8 in experiment 1 and P+8 in experiment 2.

Treatment of Cells
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about
48 hours at 37±1.0oC in the presence of 5% CO2. Initially, experiment 3 did not pass all the acceptability criteria and therefore this part of the study was repeated. In total 3 valid experiments were performed.

Luciferase Activity Measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Results and discussion

Positive control results:
The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
The EC1.5 of the positive control was between 5 and 125 µM (64 µM, 96 µM and 82 µM in experiment 1, 2 and 3, respectively). A clear dose response was observed (2.55-fold, 1.94-fold and 2.48-fold in experiment 1, 2 and 3, respectively).

In vitro / in chemico

Resultsopen allclose all
Run / experiment:
other: 1
Parameter:
other: Imax
Value:
1.18
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Remarks:
vehicle was negative control
Positive controls validity:
valid
Remarks on result:
other: No luminescence activity induction compared vehicle control
Run / experiment:
other: 2
Parameter:
other: Imax
Value:
1.75
Vehicle controls validity:
valid
Negative controls validity:
not examined
Remarks:
vehicle was negative control
Positive controls validity:
valid
Remarks on result:
other: dose-related luminescence activity was observed
Run / experiment:
other: 2
Parameter:
other: EC1.5 (µM)
Value:
331
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Remarks:
vehicle was negative control
Positive controls validity:
valid
Run / experiment:
other: 2
Parameter:
other: IC30 (µM)
Value:
1 292
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Remarks:
vehicle was negative control
Positive controls validity:
valid
Remarks on result:
other: Cellular toxicity was observed
Run / experiment:
other: 2
Parameter:
other: IC50 (µM)
Value:
1 497
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Remarks:
vehicle was negative control
Positive controls validity:
valid
Remarks on result:
other: Cellular toxicity was observed
Run / experiment:
other: 3
Parameter:
other: Imax
Value:
1.09
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Remarks:
vehicle is negative control
Positive controls validity:
valid
Remarks on result:
other: No luminescence activity induction compared vehicle control
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: cellular toxicity was noted in experiment 2 only.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (10.1%, 6.1% and 6.7% in experiment 1, 2 and 3, respectively).
- Acceptance criteria met for positive control:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was between 5 and 125 µM (64 µM, 96 µM and 82 µM in experiment 1, 2 and 3, respectively). A clear dose response was observed (2.55-fold, 1.94-fold and 2.48-fold in experiment 1, 2 and 3, respectively).

Any other information on results incl. tables

Acceptability Criteria

The KeratinoSensTM test is considered acceptable if it meets the following criteria:

a)     The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).

b)     The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol 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.

c)     Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented. 

 Data analysis

The following parameters are calculated in the KeratinoSensTM test method:

·        The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control

·        The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained

·        The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

 

Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the averageof the individual repetitions.

Equation 1: Fold induction ((Lsample - Lblank) / (Lsolvent - Lblank))

Where:

Lsample is the luminescence reading in the test chemical well

Lblank is the luminescence reading in the blank well containing no cells and no treatment

Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative) control

 

The EC1.5is calculated by linear interpolation according to Equation 2 ,and the overall EC1.5 is calculated as the mean of the individual repetitions.

Equation 2: EC1.5 = (Cb - Ca) x ((1.5 -Ia) / (Ib-Ia)) + Ca

Where:

Ca       is the lowest concentration in μM with > 1.5 fold induction

Cb       is the highest concentration in μM with < 1.5 fold induction

Ia        is the fold induction measured at the lowest concentration with > 1.5 fold induction (mean of three replicate wells)

Ib        is the fold induction at the highest concentration with < 1.5 fold induction (mean of three replicate wells)

 

Viability is calculated by Equation 3: Viability = ((Vsample - Vblank) / (Vsolvent - Vblank)) x 100

Where:

Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment

Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent (negative) control

Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.

Equation 4: ICx = (Cb - Ca) x (((100 -x)-Va) / (Vb - Va)) + Ca

x is the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)

Ca is the lowest concentration in μM (or µg/mL) with > x% reduction in viability

Cb is the highest concentration in μM (or µg/mL) with < x% reduction in viability

Va is the % viability at the lowest concentration with > x% reduction in viability

Vb is the % viability at the highest concentration with < x% reduction in viability

In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

Data Interpretation

A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTMprediction is considered negative:

1.        The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s
t-test)

2.       The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)

3.        The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)

4.       There is an apparent overall dose-response for luciferase induction

Negative results obtained with concentrations <1000 µM or 200 µg/mL should be considered as inconclusive. 

Results

2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. An overview of the viability and luciferase activity induction is summarized in Table 1. The results of the positive control are summarized in Table 2. An overview of EC1.5, Imax, IC30 and IC50 values is given in Table 3.

Three independent experiments were performed. The cells were in these experiments incubated withthe test itemin a concentration range of 0.98 – 2000 µM (2-fold dilution steps) for 48 hours. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

Experiment 1

·          No precipitation was observed at the start and end of the incubation period in the 96-well plates.

·          The test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated. 

·          No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.18 and therefore no EC1.5 could be calculated. 

·          The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.55 and the EC1.564 µM. 

Experiment 2

·          No precipitation was observed at the start and end of the incubation period in the 96-well plates.

·          The test item showed toxicity. The calculated IC30 was 1295 µM and the calculated IC50 was 1497 µM. 

·          A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 1.75 and the EC1.5 331µM.

·          The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 1.94 and the EC1.596 µM.

Experiment 3

·          No precipitation was observed at the start and end of the incubation period in the 96-well plates.

·          The test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated. 

·          No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imaxwas 1.09 and therefore no EC1.5could be calculated.

The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imaxwas 2.46 and the EC1.582 µM.

Both tests passed the acceptance criteria:

·          The luciferase activity induction obtained with the positive control,Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. 

·          The EC1.5 of the positive control was between 5 and 125 µM (64 µM, 96 µM and 82 µM in experiment 1, 2 and 3, respectively). A clear dose response was observed (2.55-fold, 1.94-fold and 2.48-fold in experiment 1, 2 and 3, respectively).

·          Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (10.1%, 6.1% and 6.7% in experiment 1, 2 and 3, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

 Concentration (µM) 0.98  2.0  3.9  7.8  16  31  63  125  250  500  1000  2000 

Exp 1 luminescence

Exp 1 viability (%)

0.99

101.3 

1.05

94.2 

1.03

104.6 

1.07

109.0 

1.13

99.1 

1.05

99.6 

1.03

104.0 

1.17

92.6 

1.18

102.8 

1.13

99.2 

1.11

96.5 

1.09

111.2 

Exp 2 luminescence

Exp 2 viability (%)

1.01

111.2 

1.09 

103.5

1.11

96.1 

1.11

94.0 

1.10

92.9

1.08

90.5

1.32

92.1

1.35

91.3 

1.42

88.8 

1.67***

94.8 

1.75***

99.3 

0.00

0.0 

Exp 3 luminescence

Exp 3 viability (%)

1.09

112.6

1.00

115.8 

1.03

107.4 

1.01

114.8 

1.04

115.0 

1.05

108.0 

0.98

107.1 

1.03

112.1 

1.00

105.1 

1.04

108.0 

0.92

97.8 

1.06

100.1 

*** p=< 0.001 Student's test

Table 2 : Overview Luminescence Induction and cell viability positive control EDMG in Experiement 1 and 2

 Concentration (µM)  7.8 16  31  63  125  250 

Exp 1 luminescence

Exp 1 viability (%)

1.07

102.4

1.15

110.8

1.28

110.8 

1.49

113.9 

1.73***

115.4 

2.55***

127.3 

Exp 2 luminescence

Exp 2 viability (%)

1.07

91.8

1.08

97.8 

1.25

97.6 

1.35

101.0 

1.63***

99.6 

1.94***

101.2 

Exp 3 luminescence

Exp 3 viability (%)

 0.84

106.5

0.97

103.3 

1.09

109.2 

1.43

110.7 

1.66***

110.1 

2.48***

104.3 

***p<0.001 Student's test

Table 3: Overview EC1.5, Imax, IC30 and IC50 Values

   EC1.5 (µM) Imax  IC30 (µM)  IC50 (µM) 
 Test Item Experiment 1 NA  1.18  NA  NA 
 Test Item Experiment 2 331  1.75  1295  1497 
 Test Item Experiment 3 NA  1.09  NA  NA 

 Pos Control Experiment 1

64  2.55  NA  NA 
 Pos Control Experiment 2 96  1.94  NA  NA 
 Pos Control Experiment 3 82  2.48  NA  NA 

NA = Not applicable

The test item showed no toxicity in experiment 1 and 3 (no IC30and IC50value), however toxicity was observed in experiment 2 at the highest test concentration only (IC30values of 1295µMand IC50values of 1497µM).No biologically relevant induction of the luciferase activity (no EC1.5value) was measured at any of the test concentrations in experiment 1.The maximum luciferase activity induction (Imax) was 1.18-fold. In experiment 2,a luminescence activity induction compared to the vehicle control was observed(EC1.5value of 331µM). The maximum luciferase activity induction (Imax) was 1.75-fold. Since the first two experiments were not concordant a third experiment was performed. In experiment 3, no luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The maximum luciferase activity induction (Imax) was 1.09-fold.

The test itemis classified as negative in the KeratinoSensTMassaysince negative results (<1.5-fold induction) were observed in 2 out of 3 experiments at test concentrations
≥ 1000µM.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
In conclusion, 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the ability of 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 449935-248 of the test item was a clear colourless liquid. The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of0.98 – 2000 µM (2-fold dilution series). The highest test concentration was thehighest dose required in the current guideline. No precipitate was observed at any dose level tested. Three independent experiments were performed

All experiments passed the acceptance criteria:

·          The luciferase activity induction obtained with the positive control,Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. 

·          The EC1.5of the positive control was between 5 and 125 µM (64 µM, 96 µM and 82 µM in experiment 1, 2 and 3, respectively). A clear dose response was observed (2.55-fold, 1.94-fold and 2.48-fold in experiment 1, 2 and 3, respectively).

·          Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (10.1%, 6.1% and 6.7% in experiment 1, 2 and 3, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

The test item showed no toxicity in experiment 1 and 3 (no IC30and IC50value), however toxicity was observed in experiment 2 at the highest test concentration only (IC30values of 1295µM and IC50values of 1497µM). No biologically relevant induction of the luciferase activity (no EC1.5value) was measured at any of the test concentrations in experiment 1.The maximum luciferase activity induction (Imax) was 1.18-fold. In experiment 2, a luminescence activity induction compared to the vehicle control was observed(EC1.5value of 331µM). The maximum luciferase activity induction (Imax) was 1.75-fold. Since the first two experiments were not concordant a third experiment was performed. In experiment 3, no luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The maximum luciferase activity induction (Imax) was 1.09-fold.

The test item is classified as negative in the KeratinoSensTMassaysince negative results (<1.5-fold induction) were observed in 2 out of 3 experiments at test concentrations ≥ 1000µM.