Registration Dossier

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Administrative data

Description of key information

An intelligent testing strategy for skin senstisation has been followed. The following studies/investigations have been performed:

- OECD 422C - DPRA study. Performed under GLP with no deviations from OECD TG.

- OECD 422D - KeratinoSens study. Performed under GLP with no deviation from OECD TG.

- QSAR - DEREK NEXUS version 6.0.1

In addition the following study is underway:

- OECD 422 - U-SENS study. Draft report due 13 May 2018

The endpoint will be addressed by a weight of evidence.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
DEREK NEXUS

2. MODEL (incl. version number)
DEREK NEXUS version 6.0.1
NEXUS: 2.2.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
C(C(OC(C(F)(F)F)C(F)(F)F)=O)(C)=C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF or providing a link]
- Defined endpoint: 4.Human health effects 4.6. Skin sensitisation
- Unambiguous algorithm: [1] Structural alerts, [2] logic of argumentation, [3] nearest neighbours (within same alert as query compound) based on Tanimoto similrity, and [4] feature-based database search
- Defined domain of applicability:
- Appropriate measures of goodness-of-fit and robustness and predictivity:
- Mechanistic interpretation:

5. APPLICABILITY DOMAIN
[Explain how the substance falls within the applicability domain of the model]
- Descriptor domain: The scopes of the structure-activity relationships describing the skin sensitation endpoint are defined by the developer to be the applicability domain for the model. Thereforeif a chemical activates an alert describing a structure-activity for skin sensitisation it can be considered to be within the applicability domain. The applicability of potency predictions may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound does not activate an alert or reasoning rule then Derek makes a negative prediction. The applicability of the negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features.
- Structural and mechanistic domains: The aplicability domain of each alert is defined by the aler developer on the basis of the training set data and expert judgement on the chemical and biological factors which affect the mechanism of action for each alert. For potency predictions, at least three nearest neighbours are required within alerting space to make a prediction. For non-alerting compounds, users should determine the applicability of negative predictions by evaluating the information supplied by Derek (i.e. the presence or absence of misclassified and/or unclassified features).
- Similarity with analogues in the training set:
- Other considerations (as appropriate):

6. ADEQUACY OF THE RESULT
Non-propietary elements of the training set are available through the references, and ilustrated by the examples, within Derek Nexus. The illustrative examples are not available, due to the proprietary nature of Derek Nexus.
External validation is carried out on each knowledge based release. The data sets used for validation are available in the public domain, but the curated versions used at Lhasa are proprietary, so are not made available.
Three published data sets have been used ofr alert validation: [1] Cronin and Basketter, [2] Gerberick et al and Kern et al and [3] a collection of local lymph node assay for 137 compounds published in Contact Dermatitis which have been extracted from Vitic Nexus (13 September 2012). Furthermore, the relationship between likelihood levels and prediction accuracy has been assessed (Judsen et al 2013). Finally, several external evaluations have been published [Rorije et al, Nukada et al].
Guideline:
other: REACH Guidance on QSARs R.6
Principles of method if other than guideline:
- Software tool(s) used including version: DEREK NEXUS version 6.0.1
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'
Justification for non-LLNA method:
The objective of this study was to obtain a prediction on the potential for skin sensitization of the test item with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used. This data is to be used as part of a weight of evidence for the endpoint skin sensitisation in order to avoid animal testing.
Specific details on test material used for the study:
SMILES:
C(C(OC(C(F)(F)F)C(F)(F)F)=O)(C)=C
Run / experiment:
other: n/a
Parameter:
other: EC3 (%)
Value:
90
Remarks on result:
other: weak indication of skin sensitisation

The result as generated by DEREK NEXUS is presented in Appendix 2. The relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) are presented in Appendix 3.

DEREK NEXUS version 6.0.1 yielded an alert for 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate for skin sensitization based on the presence of the alpha,beta-unsaturated ester group. 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate is predicted to be sensitizing to the skin (plausible). DEREK NEXUS predicts an EC3 of 90% (weak sensitizer) for the alpha,beta-unsaturated ester group based on data on closest structurally-related substances. The mechanism for the alert is thought to occur through Michael addition.

Interpretation of results:
study cannot be used for classification
Conclusions:
The result as generated by DEREK NEXUS is presented in the attached document 'DEREK prediction report'. The relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) are attached under 'attached justification'.
DEREK NEXUS version 6.0.1 yielded an alert for 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate for skin sensitization based on the presence of the alpha,beta-unsaturated ester group. 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate is predicted to be sensitizing to the skin (plausible). DEREK NEXUS predicts an EC3 of 90% (weak sensitizer) for the alpha,beta-unsaturated ester group based on data on closest structurally-related substances. The mechanism for the alert is thought to occur through Michael addition.
The outcome of the assessment is that the substance should not be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.
Executive summary:

Introduction

The objective of this study was toobtain a prediction on the potential for skin sensitization of the test item with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

Background/Scope

DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitization based on the presence of molecular substructures. LHASA has inserted validation comments for the skin sensitization alerts.

 

The level of likelihood of a structure being sensitizing to skin is expressed in terms of:

Certain                   There is proof that the proposition is true.

Probable     There is at least one strong argument that the proposition is true and there are no arguments against it.

Plausible    The weight of evidence supports the proposition.

Equivocal   There is an equal weight of evidence for and against the proposition.

 

The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which are evaluated as being equivocal or greater was used in this assessment. 

 

If a substance is predicted to be no skin sensitizer, DEREK NEXUS contains an expert-derived functionality to provide negative predictions for skin sensitization. This functionality further evaluates those compounds which do not fire any skin sensitization alerts in DEREK NEXUS. The query compound is compared to a Lhasa reference set of Ames test or skin sensitization data, producing the following outcomes:

•          In compounds where all features in the molecule are found in accurately classified compounds from the reference set, a negative prediction is displayed: inactive.

•          For those query compounds where features in the molecule are found in non-alerting skin sensitizers in the Lhasa reference set, the prediction remains negative and the misclassified[1]features are highlighted to enable the negative prediction to be verified by expert assessment.

          In cases where features in the molecule are not found in the Lhasa reference set, the prediction remains negative and the unclassified[2]features are highlighted to enable the negative prediction to be verified by expert assessment.

 

If a substance is predicted to be a skin sensitizer, its potency is predicted by DEREK NEXUS by calculating an EC3 value based on experimental data from the closest structurally-related substances (at least 3 substances should be present) using the following equation:

EC3Q= MWQ /(Σ ωNN/ Σ TNN)

MW = molecular weight

T = Tanimoto similarity score

ω = weighting factor = (MWNN/EC3) * TNN

Q = query compound

NN = nearest neighbour

 

The EC3 is the estimated concentration needed to produce a stimulation index of 3.

Results and Conclusion

The result as generated by DEREK NEXUS is presented in Appendix 2. The relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) are presented in Appendix 3.

DEREK NEXUS version 6.0.1 yielded an alert for 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate for skin sensitization based on the presence of the alpha,beta-unsaturated ester group. 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate is predicted to be sensitizing to the skin (plausible). DEREK NEXUS predicts an EC3 of 90% (weak sensitizer) for the alpha,beta-unsaturated ester group based on data on closest structurally-related substances. The mechanism for the alert is thought to occur through Michael addition.


[1]Unclassified features are those that have not been found in the Lhasa Ames test reference set


[2]Misclassified features are those that have been derived from non-alerting mutagens in the Lhasa Ames test reference set

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
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).
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
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.
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).
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).

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.

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.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
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 the refrigerator (2-8ºC)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Final dilution of a stock liquid: 100mM solutions

Details on the study design:
Skin sensitisation (In chemico test system) - Details on study design:
Test system: Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA COOH) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.
Rationale: Recommended test system in the international OECD guideline for DPRA studies.
Source: JPT Peptide Technologies GmbH, Berlin, Germany
Storage: The peptides were stored in the freezer (≤-15ºC) for a maximum of 6 months
Positive control results:
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 76.5% ± 0.7%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 56.0% ± 0.2%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).
Run / experiment:
other: mean value
Parameter:
other: mean SPCC depletion (%)
Value:
100
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: mean value
Parameter:
other: mean SPCL depletion (%)
Value:
9.1
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: mean value
Parameter:
other: mean SPCC / SPCL depletion (%)
Value:
54.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Solubility Assessment of the Test Item

At a concentration of 100 mM, 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate was not soluble in MQ and ACN/MQ (1:1, v/v), but was soluble in ACN and isopropanol. Since the use of ACN is preferred, this solvent was used to dissolvethe test itemin this DPRA study.

Cysteine Reactivity Assay

The reactivity of 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC-PDA analysis, following 24 hours of incubation at 25±2.5°C. Representative chromatograms of CCcys-209029/A and 209029/A-cys samples are presented in Appendix 4. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 3 (Appendix 3).

Acceptability of the Cysteine Reactivity Assay

The SPCC standard calibration curve is presented in Figure 1 (Appendix 2). The correlation coefficient (r2) of the SPCC standard calibration curve was 0.995. Since the r2was >0.99, the SPCC standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented in Table 4 (Appendix 3). The mean peptide concentration of Reference Controls A was 0.500 ± 0.004 mM while the mean peptide concentration of Reference Controls C was 0.505 ± 0.005 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolvethe test item did not impact the Percent SPCC Depletion.

The SPCC peak areas for Reference controls B and C are presented in Table 5 (Appendix 3). The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.0%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCC A220/A258 area ratios of Reference controls A, B and C are presented in Table 6 (Appendix 3). The mean area ratio (A220/A258) of the Reference Control samples was 18.70. The mean A220/A258 ratio ± 10% range was 16.83-20.57. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

The results of the positive control cinnamic aldehyde are presented in Table 7 (Appendix 3). The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 76.5% ± 0.7%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Results Cysteine Reactivity Assay for the Test Item

Preparation of a 100 mM 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. No precipitate was observed in any of the samples. 

The results of the cysteine reactivity assay for the test item are presented in Table 8 (Appendix 3). In the CC sample no peak was observed at the retention time of SPCC (see chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item with SPCC. For the 209029/A-cys samples, the mean SPCC A220/A258 area ratio could not be calculated, since the test item displayed high reactivity towards SPCC. Overall, it can be concluded that the test item did not co-elute with SPCC.

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test item was 100.0% ± 0.0%.

Lysine Reactivity Assay

Two experiments to determine the reactivity of the test item towards SPCL were performed. During the first experiment which was performed on the 08thof January 2017, the SPCL calibration curve did not meet the acceptance criteria (r2<0.99) and therefore the results of this experiment were not accepted. The results will be included in the raw data files of the study but will not be reported. The experiment was repeated on the 16thof January 2018 and the results of this experiment are reported below.

The reactivity of 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis, following 24.5 hours of incubation at 25±2.5°C. Representative chromatograms of CClys-209029/A and 209029/A-lys samples are presented in Appendix 4. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 9 (Appendix 3).

Acceptability of the Lysine eactivity Assay

The SPCL standard calibration curve is presented in Figure 2 (Appendix 2). The correlation coefficient (r2) of the SPCL standard calibration curve was 0.996. Since the r2was >0.99, the SPCL standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented in Table 10 (Appendix 3). The mean peptide concentration of Reference Controls A was 0.511 ± 0.013 mM while the mean peptide concentration of Reference Controls C was 0.501 ± 0.006 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.

The SPCL peak areas for Reference controls B and C are presented in Table 11 (Appendix 3). The CV of the peptide areas for the nine Reference Controls B and C was 1.3%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCL A220/A258 area ratios of Reference controls A, B and C are presented in Table 12 (Appendix 3). The mean area ratio (A220/A258) of the Reference Control samples was 15.03. The mean A220/A258 ratio ± 10% range was13.52-16.53. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

The results of the positive control cinnamic aldehyde are presented in Table 13 (Appendix 3). The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 56.0% ± 0.2%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Results Lysine Reactivity Assay for the Test Item

Preparation of a 100 mM 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. Upon preparation a precipitate was observed in the CC and the test item samples. After incubation no precipitate was observed in any of the samples.

The results of the lysine reactivity assay for the test item are presented in Table 14 (Appendix 3). In the CC sample no peak was observed at the retention time of SPCL (see chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item with SPCL. For the209029/A-lys samples, the mean SPCL A220/A258 area ratio was 15.46. Since this was within the 13.52-16.53 range, this again indicated that there was no co-elution ofthe test itemwith SPCL. 

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the Test Item was 9.1% ± 0.9%.

DPRA Prediction and Reactivity Classification

Upon preparation as well as after incubation of the SPCC test item samples, no precipitate was observed in the samples. Upon preparation of the SPCL test item samples, a precipitate was observed. After incubation of the SPCL test item samples, no precipitate was observed in the samples.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 100.0% SPCC depletion while in the lysine reactivity assay the test item showed 9.1% SPCL depletion. The mean of the SPCC and SPCL depletion was 54.5% and as a result the test item was positive in the DPRA and was classified in the “highreactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Therefore, the test item was considered to be positive in the DPRA.

      SPCC depletion SPCL depletion     Mean of SPCC and SPCL depletion  DPRA prediction and reactivity classification 
 Test Item Mean   ± SD Mean  ± SD   Cysteine 1:10 / Lysine 1:50 prediction model 
 2,2,2 -trifluoro-1 -(trifluoromethyl)ethyl methacrylate 100.0%  ± 0.0% 9.1%  ± 0.9% 54.5%  Positive: High reactivity 
Interpretation of results:
study cannot be used for classification
Conclusions:
In conclusion, this DPRA test is valid. 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Justification for classification or non-classification

A weight of evidence approach is applied for the assessment of skin sensitising properties of ,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate. At present, the following data are available:

- A DEREK assessment that concludes there is a structural alert for skin sensitising properties (presence of an alpha,beta-unsaturated ester group).

-The DPRA was positive (100.0% SPCC depletion, 9.1% SPCL depletion)

-The Keratinosense study was negative.

As the results of all three studies are not in agreement a third in vitro test (the U-Sens assay) is required and is underway.

 

It is therefore not possible to conclude on the classification and labelling at this point. The dossier will be updated once the additional data is available.