1,3-Dioxole, 2,2,4-trifluoro-5-(trifluoromethoxy)

Endpoint summary

Description of key information

DEREK NEXUS version 5.0.2 (CRL, 2017, K1) : inconclusive

in vitro DPRA test (CRL, 2017, K1): positive

in vitro KeratinoSens test (CRL, 2017, K1): positive

Based on a weight-of-evidence approach, the substance is considered as skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18-Jan-2017 to 07-Mar-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 442C.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

04 February 2015

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Assessed on 07-11, 14 and 16 September 2015. Dated on the 03 November 2015.

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Test item preparation:
A correction factor of 1.044 was applied for the purity of the test item.
Solubility of the test item was assessed before performing the DPRA assay. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvent was evaluated: acetonitrile (ACN).
Perfluoro methoxy dioxole stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay, 985.9 μL of cold ACN was pipetted into an amber glass vial and 14.1 μL of ice-cold Perfluoro methoxy dioxole was added. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The 100 mM test item solution in ACN was directly used for sample preparation and was kept at room temperature in a closed amber vial during this time. The test item, positive control and peptide samples were prepared less than 50 minutes before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assays.

Details on the study design:

See section "Any other information on materials and methods incl. tables"

Positive control results:

Cinnamic aldehyde was used as a positive control.

- Result of positive control samples in cysteine assay: Mean of SPCC depletion = 72.0% and SD = 0.8%
- Result of positive control samples in lysine assay: Mean of SPCL depletion = 43.5% and SD = 2.1%

The mean percent peptide depletion values for the positive control with its standard deviation value were within the acceptability criteria for the DPRA assay (cysteine and lysine reactivity assays).

Key result

Parameter:

other: Mean SPCC and SPCL depletion

Remarks:

(%)

Value:

45.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Reference controls

Positive controls validity:

valid

Remarks on result:

other: High reactivity

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not applicable

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Not specified

Acceptability of the cysteine reactivity assay

The correlation coefficient (r2) of the SPCC standard calibration curve was 0.995. Since the r2 was >0.990, the SPCC standard calibration curve was accepted.

The mean peptide concentration of Reference Controls A was 0.534 ± 0.011 mM while the mean peptide concentration of Reference Controls C was 0.534 ± 0.009 mM. The mean 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 Perfluoro methoxy dioxole did not impact the Percent SPCC Depletion.

The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.9%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The mean area ratio (A220/A258) of the Reference Control samples was 18.33. The mean A220/A258 ratio ± 10% range was 16.50-20.16. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Acceptability of the lysine reactivity assay

The correlation coefficient (r2) of the SPCL standard calibration curve was 0.994. Since the r2 was >0.990, the SPCL standard calibration curve was accepted.

The mean peptide concentration of Reference Controls A was 0.492 ± 0.007 mM while the mean peptide concentration of Reference Controls C was 0.482 ± 0.007 mM.

The mean 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 Perfluoro methoxy dioxole did not impact the Percent SPCL Depletion.

The CV of the peptide areas for the nine Reference Controls B and C was 1.9%. This was within the acceptance criteria (CV <15.0%) and confirmed the stability of the HPLC run over time.

The mean area ratio (A220/A258) of the Reference Control samples was 13.98. The mean A220/A258 ratio ± 10% range was 12.58-15.38. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

SPCC and SPCL depletion and reactivity classification for the test substance

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and	Reactivity class
	Mean	+/- SD	Mean	+/- SD	SPCL depletion	Cysteine 1:10 / Lysine 1:50 prediction model
	83.4%	+/- 3.6%	8.4%	+/- 3.4%	45.9%	High reactivity

Interpretation of results:

other: High reactivity

Conclusions:

Perfluoro methoxy dioxole was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The skin sensitisation potential of Perfluoro methoxy dioxole was evaluated using an in chemico direct peptide binding assay (DPRA) according to the OECD Guideline No. 442C and in compliance with GLP.

Following 24 hours of incubation of Perfluoro methoxy dioxole with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for Perfluoro methoxy dioxole were all within the acceptability criteria for the DPRA assay.

No co-elution of the test item with SPPC or SPCL was observed.

In the cysteine reactivity assay Perfluoro methoxy dioxole showed 83.4% SPCC depletion while in the lysine reactivity assay Perfluoro methoxy dioxole showed 8.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 45.9% and as a result Perfluoro methoxy dioxole was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Therefore, Perfluoro methoxy dioxole was considered to be positive in the DPRA.

It can be concluded that this DPRA test is valid.

Perfluoro methoxy dioxole was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Reference 2

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

08 February 2017

Reliability:

2 (reliable with restrictions)

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: Nexus: 2.1.1

2. MODEL (incl. version number): Derek Nexus: 5.0.2

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
- Smiles: C1(OC(=C(O1)F)OC(F)(F)F)(F)F
- Average Mol Mass: 210.03
- Exact Mol Mass: 209.9752
- Log Kp: -1.87
- Log P: 3

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See QMRF attached (see attached full study report)

5. APPLICABILITY DOMAIN
See QPRF attached (see attached full study report)

6. ADEQUACY OF THE RESULT
The assessment of perfluoro methoxy dioxole using DEREK NEXUS could not predict the skin sensitizer potential of the substance and therefore the assessment was considered to be inconclusive due to the absence of fluorated substances in its database.

Qualifier:

no guideline required

Principles of method if other than guideline:

Derek Nexus is a proprietary, rule-based expert system for the prediction of toxicity.

GLP compliance:

no

Justification for non-LLNA method:

This information was obtained according to requirements for skin sensitization of Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the relevant classification and/or risk assessment obligations. The weight of evidence approach according to Annex XI, sections 1.2-1.5, to the REACH Regulation was used.

Parameter:

other: DEREK NEXUS 5.0.2.

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: Not relevant; DEREK NEXUS did not yield any alerts for skin sensitization.

Interpretation of results:

GHS criteria not met

Conclusions:

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. However, the assessment using DEREK Nexus could not predict the skin sensitizer potential of the substance due to the absence of fluorated substances in its database. Therefore the assessment was considered to be inconclusive.

Executive summary:

DEREK NEXUS 5.0.2. software was used to predict the skin sensitization potential of perfluoro methoxy dioxole.

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item.

Perfluoro methoxy dioxole is predicted to be not sensitizing to the skin.

However, the assessment using DEREK Nexus could not predict the skin sensitizer potential of the substance due to the absence of fluorated substances in its database. Therefore the assessment was considered to be inconclusive.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 January 2017-01 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 442D. Furthermore, functional model conditions and references to historical control data are included in the report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

Adopted February, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Assessed on 07-11, 14 and 16 September 2015. Dated on the 03 November 2015.

Type of study:

other: KeratinoSens assay (inflammatory response in keratinocytes)

Specific details on test material used for the study:

Test item preparation:
A correction factor of 1.043 was used to correct for the composition/purity of the test item.
The test item was dissolved in DMSO to a final concentration of 200 mM. The compound formed a clear colourless solution at 200 mM. From the stock 11 spike solutions in DMSO were prepared (2-fold dilution series): 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, 0.391, 0.195 and 0.0977 mM. The stock and spike solution were diluted 25-fold with exposure medium (final concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95 and 0.977 μM (final concentration DMSO of 1%).

Since the test item was very volatile, all test item solutions were kept on ice until addition to the test system. This was done for all steps except the preparation of the DMSO stocks and spike solutions in 100% DMSO since 100% DMSO solidifies when it is kept on ice. The test item concentrations were added to the test system as soon as possible after preparation.

Details on the 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).

Cell culture:
- Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.
- Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 μg/ml).
- Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 65 – 93 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 – 37.0°C).

Preparation of the positive control:
The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol, for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 5.1, so that the final concentration of the positive control ranges from 7.81 to 250 μM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

Subculturing:
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages.

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.

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 substances 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. In total 3 independent experiments were performed.
Care was taken to avoid evaporation of the volatile test compound. Therefore in the present study wells containing the test item were covered with a seal during compound incubation.

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 luminometer to assess the quantity of luciferase (integration time one second).

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 with the TECAN Infinite® M200 Pro Plate Reader.

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.

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 KeratinoSensTM prediction is considered negative (See section "Illustration"):
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 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.

Acceptance criteria:
- 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 7.81 to 250 μM).
- 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.
- 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.

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Remarks:

EC1.5 = 622 µM

Value:

2.32

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Remarks:

EC1.5 = No calculation possible

Value:

1.25

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 3

Parameter:

other: Imax

Remarks:

EC1.5 = 5.44 µM

Value:

4.6

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not applicable

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF 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 in experiment 2 and 3 (36.8 and 25.3 μM in experiment 2 and 3, respectively). The EC1.5 in the first experiment was 229 μM, just above 125 μM. In all experiments, a dose response of the positive control was observed and the induction at 250 μM was higher than 2-fold in experiment 2 and 3 (3.50-fold and 4.21-fold in experiment 2 and 3, respectively). The induction at 250 μM in experiment 1 was 1.52, below 2-fold. The effect of the positive control in experiment 1 was therefore not completely according the acceptance criteria. Although the positive control in experiment 1 showed a slightly lower effect than required according the acceptance citeria the data of the experiment 1 could be used, since Perfluoro methoxy dioxole showed a clear positive effect.
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (11%, 8.3% and 17% in experiment 1, 2 and 3 respectively).

Three independent experiments were performed. The cells were in these experiments incubated with Perfluoro methoxy dioxole in a concentration range of 0.977 – 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

- Perfluoro methoxy dioxole 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.

- A luminesce activity induction compared to the vehicle control was observed after treatment with Perfluoro methoxy dioxole . The Imax was 2.32 and the EC1.5 622 μM

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 1.52 and the EC1.5 229 μM.

Experiment 2

- Perfluoro methoxy dioxole 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 luminesce activity induction compared to the vehicle control was observed at any of the test concentrations. The Imax was 1.25 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 3.50 and the EC1.5 36.8 μM.

Experiment 3

- Perfluoro methoxy dioxole 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.

- A luminesce activity induction compared to the vehicle control was observed after treatment with Perfluoro methoxy dioxole . The Imax was 4.60 , the EC1.5 5.44 μM

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 4.21 and the EC1.5 25.3 μM.

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

The test item showed no toxicity (no IC30 and IC50 value). A statistically significant induction of the luciferase activity (EC1.5 value 622 μM; p<0.001 Student’s t test) was measured in the first experiment. The maximum luciferase activity induction (Imax) was 2.32-fold at 2000 μM.

In the second experiment no induction of the luciferase activity (no EC1.5 value) was measured. The maximum luciferase activity induction (Imax) was 1.25-fold. Since the first two experiments obtained different results a third experiment was performed. In the third experiment, a statistically significant induction of the luciferase activity (EC1.5 value 5.44 μM; p<0.001 Student’s t test) was measured. The maximum luciferase activity induction (Imax) was 4.60-fold.

The variation between the two EC1.5 values in experiment 1 and 3 are relatively large (622 μM and 5.44 in experiment 1 and 3, respectively). This may have been caused by the fact that the compound is very volatile. In addition it may be the reason for the negative effect in experiment 2. This does not affect the conclusion of the present study. However care should be taken to use the present EC1.5 data for potency assessment.

Overall, Perfluoro methoxy dioxole is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations of < 1000 μM with a cell viability of >70% compared to the vehicle control in two out of three experiments.

Finally, it is concluded that the KeratinoSensTM assay is valid and that Perfluoro methoxy dioxole is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Table 7.4.1/1. Overview luminescence induction and cell viability Perfluoro methoxy dioxole in Experiment 1, 2 and 3

Concentration (µM)	0.977	1.95	3.91	7.81	15.6	31.3	62.5	125	250	500	1000	2000
Exp 1 luminescence Exp 1 viability (%)	1.02 97.4	0.94 93.3	1.04 98.6	1.04 88.8	1.01 87.5	0.98 94.4	1.01 94.8	1.06 92.9	1.07 98.0	1.34 99.4	2.01*** 108.1	2.32*** 116.3
Exp 2 luminescence Exp 2 viability (%)	0.99 114.6	1.25 103.5	1.15 90.0	1.17 95.5	1.04 86.6	1.07 89.1	1.01 98.5	1.06 92.1	1.01 94.1	0.99 99.0	0.86 93.2	0.89 96.0
Exp 3 luminescence Exp 3 viability (%)	1.12 99.0	1.34 88.3	1.45 91.9	1.57*** 96.1	1.67*** 95.8	1.73*** 97.1	1.95*** 97.8	2.13*** 105.5	2.25*** 111.4	2.82*** 108.5	2.86*** 117.5	4.60*** 115.9

*** P<0.001 Student's t test

Table 7.4.1/2. Overview luminescence induction and cell viability positive control Ethylene dimethacrylate glycol in Experiment 1, 2 and 3

Concentration (µM)	7.81	15.6	31.3	62.5	125	250
Exp 1 luminescence Exp 1 viability (%)	0.81 123.6	0.85 130.6	0.89 133.2	1.06 146.9	1.22 148.1	1.52*** 146.0
Exp 2 luminescence Exp 2 viability (%)	1.04 96.4	1.22 92.2	1.46 90.1	1.71*** 111.0	2.23*** 111.4	3.50*** 108.9
Exp 3 luminescence Exp 3 viability (%)	1.18 96.7	1.40 97.8	1.56*** 105.2	2.21 113.0	2.82*** 114.8	4.21*** 123.3

*** P<0.001 Student's t test

Table 7.4.1/3. Overview EC_1.5, I_max, IC₃₀ and IC₅₀ values

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test item Experiment 1	622	2.32	NA	NA
Test item Experiment 2	NA	1.25	NA	NA
Test item Experiment 3	5.44	4.60	NA	NA
Pos Control Experiment 1	229	1.52	NA	NA
Pos Control Experiment 2	36.8	3.50	NA	NA
Pos Control Experiment 3	25.3	4.21	NA	NA

NA = Not applicable

Interpretation of results:

other: Positive response

Conclusions:

Perfluoro methoxy dioxole is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) in the KeratinoSensTM assay under the experimental conditions described in the report.

Executive summary:

Perfluoro methoxy dioxole was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in a KeratinoSens(TM) test according to the OECD Guideline No. 442D and in compliance with GLP.

In this test, KeratinoSens(TM) cells cultures were treated with 50µL of the 25 -fold diluted test chemical (0.977, 1.95, 3.91, 7.81, 15.6, 62.5, 125, 250, 500, 1000 and 2000 µM) and control substances (positive control: ethylene dimethacrylate glycol in DMSO, vehicle control: 1% DMSO in exposure medium, blank) and were then incubated for about 48 hours at 37 +/-1.0°C in presence of 5% CO2. Luciferase activity (Imax and EC1.5) was assessed using a luminometer. The toxicity of the test substance was evaluated with the assessment of cell viability (MTT test). A total of 3 independent experiments were performed. Care was taken to avoid evaporation of the volatile test compound.

The test item showed no toxicity (no IC30 and IC50 value).

A statistically significant induction of the luciferase activity (EC1.5 value 622 μM; p<0.001 Student’s t test) was measured in the first experiment. The maximum luciferase activity induction (Imax) was 2.32-fold at 2000 μM.

In the second experiment no induction of the luciferase activity (no EC1.5 value) was measured. The maximum luciferase activity induction (Imax) was 1.25-fold. Since the first two experiments obtained different results a third experiment was performed.

In the third experiment, a statistically significant induction of the luciferase activity (EC1.5 value 5.44 μM; p<0.001 Student’s t test) was measured. The maximum luciferase activity induction (Imax) was 4.60-fold.

Overall, Perfluoro methoxy dioxole is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations of < 1000 μM with a cell viability of >70% compared to the vehicle control in two out of three experiments. Finally, it is concluded that the KeratinoSensTM assay is valid.

Perfluoro methoxy dioxole is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) in the KeratinoSensTM assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A Weight-of-Evidence approach has been used to fulfill information requirements about skin sensitisation potential of perfluoro methoxy dioxole.

The weight of evidence approach was used according to Annex XI, sections 1.2-1.5, to the REACH Regulation.

The three following studies were used:

- DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item, and perfluoro methoxy dioxole is predicted to be not sensitizing to the skin. DEREK does not seem to have fluorated substances in its database, because if the fluor atoms are replaced by hydrogens and DEREK is run for methoxy dioxole, DEREK recognizes methoxy dioxole as a precursor for an aldehyde – with formation of formaldehyde – and the substance is considered positive. Therefore, the assessment using DEREK Nexus could not predict the skin sensitizer potential of the perfluoro methoxy dioxole probably due to the absence of fluorated substances in its database. Therefore the assessment was considered to be inconclusive.

- A valid Direct Peptide Reactivity Assay (DPRA) was performed according to OECD 442C and in agreement with GLP principles. In the cysteine reactivity assay the test item showed 83.4% SPCC depletion, and in the lysine reactivity assay the test item showed 8.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 45.9%. As a result, perfluoro methoxy dioxole was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model and was considered to be positive in the DPRA.

- A valid KeratinoSensTM assay was performed according to OECD 442D and in agreement with GLP principles. The test item showed no toxicity. The maximum luciferase activity induction (Imax) was 2.32-fold at 2000 μM in experiment 1. In experiment 2 no induction of the luciferase activity was measured: 1.25-fold at 1.95 μM decreasing to 0.89-fold at 2000 μM. Since in the first two experiments different results were obtained, a third experiment was performed. In the third experiment, a statistically significant induction of the luciferase activity (EC1.5 value 5.44 μM) was measured. The maximum luciferase activity induction was 4.60-fold. Overall, perfluoro methoxy dioxole is classified as positive in the KeratinoSensTM assay, since positive results (>1.5-fold induction) were observed at attest concentrations of <1000 μM with a cell viability of >70% compared to the vehicle control in two out of three experiments.

This information was obtained according to requirements for skin sensitization of Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the relevant classification and/or risk assessment obligations.

Interpretation and conclusion of the Weight-of-Evidence:

The assessment of perfluoro methoxy dioxole using DEREK NEXUS could not predict the skin sensitizer potential of the substance and therefore the assessment was considered to be inconclusive.

The DPRA assay and the KeratinoSensTM assay performed with perfluoro methoxy dioxole were both positive.

Based on all the mentioned data, the substance is considered to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonized classification:

The substance does not have an harmonized classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

According to EU CLP regulation (Consolidated version 01 -01 -2017), skin sensitisers shall be classified in category 1 where data (human or animal data) are not sufficient for sub-categorisation. Current available data are in vitro/in chemico tests (DEREK Nexus assessment, in vitro DPRA test and in vitro KeratinoSens test). There is currently no agreed strategy on how to use the results of these methods for potency assessment.

In the absence of sufficient data for sub-categorisation, the substance is proposed to be classified as Skin sensitiser Category 1.