1,2-dichloro-1,1,2-trifluoro-2-(trifluoromethoxy)ethane

Endpoint summary

Description of key information

A DEREK assessment, Direct Peptide Reactivity Assay (DPRA) and KeratinoSensTM assay were performed in accordance with 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 strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a. (v.6.0 July 2017).

A DEREK prediction on the skin sensitizing potential of Methylic Adduct was negative.

Methylic Adduct did not react with cysteine and lysine containing peptides in the DPRA thereby concluding the DPRA as negative. The KeratinoSensTM assay did not show a biologically relevant activation of keratinocytes when exposed to Methylic Adduct and was therefore negative.

Based on the current data it is concluded that Methylic Adduct does not have skin sensitizing properties. Therefore, it does not need to be classified as skin sensitizer according to Regulation (EC) No 1272/2008 (CLP) and related amendments.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Remarks:

DEREK assessment

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

02 Augustus 2018

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:

An in silico assesment is mentioned in the ECHA guidance as one of the non-animal data that may be provided as weight of evidence.

Principles of method if other than guideline:

An in silico assessment was performed with DEREK NEXUS version 6.0.1.

GLP compliance:

no

Justification for non-LLNA method:

Since 11 October 2016 it is legally required to consider all available information for the endpoint skin sensitisation and to use a non in vivo test strategy based on in chemico, in silico and in vitro skin tests combined with a WoE. An in vivo test (LLNA) is only allowed as last resort. DEREK results are adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitisation.

Key result

Run / experiment:

other: Methylic Adduct

Parameter:

other: Prediction on skin sensitization

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Methylic Adduct does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer.

Interpretation of results:

study cannot be used for classification

Remarks:

Study is part of a weight of evidence approach and is not used for classification on its own

Conclusions:

In a DEREK assessment Methylic Adduct did not yield any structual alerts for skin sensitization and was predicted to be not sensitizing to the skin.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 September 2018 - 18 September 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The validated in chemico skin sensitization test is the DPRA, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of a weight of evidence approach.

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)

Justification for non-LLNA method:

A validated in chemico skin sensitization test is the DPRA, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as one of the non-animal tests to be performed as part of a weight of evidence approach.

Details on the study design:

TEST ITEM PREPARATION
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. 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 solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol, acetone:ACN (1:1, v/v) and dimethylsulfoxide (DMSO):ACN (1:9, v/v), ethanol and methanol. Test item stock solutions were prepared freshly for each reactivity assay.

For both the cysteine and lysine reactivity assay 42.05 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1775 μL ACN after vortex mixing to obtain a 100 mM solution. After preparation, the 100 mM test item solution was kept on ice in a closed vial until preparation of the test item samples in order to prevent evaporation of the test item. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

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.
Incubation: AAfter preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.5 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation

POSITIVE CONTROL
Cinnamic aldehyde
- Purity: 99.1%
- Batch: MKCB9907
- Expiry of batch: 30 November 2021

DATA EVALUATION:
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion= [1-(Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))]x100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%
DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test substance. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see Table 1), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r^2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.

The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: Mean SPCC depletion(%)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 2.7%

Positive controls validity:

valid

Remarks:

Mean percentage SPCC: 70.3% ± 0.4%.

Remarks on result:

other: SD: 0.0%

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: Mean SPCL depletion(%)

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 2.1%

Positive controls validity:

valid

Remarks:

Mean percentage SPCL: 48.6% ± 0.7%.

Remarks on result:

other: SD: 0.3%

Other effects / acceptance of results:

Upon preparation of the SPCL test item samples, a precipitate was observed, however, after incubation no precipitate or phase separation was observed in any of the samples.

All acceptability criteria were met and therefore the DPRA was considered to be valid.
See Table 2 & 3 in "any other information on results incl. tables" for data on acceptibility criteria.

Table 2: Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.997	>0.99	0.998
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.518 ± 0.003	0.50 ± 0.05	0.508 ± 0.018
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.514 ± 0.007	0.50 ± 0.05	0.518 ± 0.006
CV (%) for RC samples B and C	<15.0	2.7	<15.0	2.1
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	70.3	40.2-69.0	48.6
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.4	<11.6	0.7
SD of peptide depletion for the test item (%)	<14.9	0.0	<11.6	0.3

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

Table 3: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
METHYLIC ADDUCT	0.0%	±0.0%	0.2%	±0.3%	0.1%	Negative: No or minimal reactivity

SD = Standard Deviation.

            

Interpretation of results:

other: Study cannot be used for classification independently, but in a WoE for the end point Skin Sensitisation.

Conclusions:

Methylic Adduct was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

In an in chemico study, performed according to OECD guideline 442C and GLP principles, the reactivity of methylic adduct towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined to assign the test chemical to one of four reactivity classes used to support the discrimination between skin sensitisers and non skin sensitisers.

Following incubation of the test substance 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 the prediction model.

ACN was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. Cinnamic aldehyde was used as a positive control. 

Upon preparation the SPCL test item samples, a precipitate was observed, however, after incubation no precipitate or phase separation was observed in any of the samples.

All validation parameters were within the acceptability criteria for the DPRA assay. Therefore, the study was considered valid.

In the cysteine reactivity assay the test item showed 0.0% SPCC depletion while in the lysine reactivity assay the test item showed 0.2% SPCL depletion. The mean of the SPCC and SPCL depletion was

0.1% and as a result methylic adduct was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 October 2018 - 19 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

One of the validated in vitro skin sensitization tests is the KeratinoSens™ assay, which is recommended in international guidelines (e.g. OECD 442D) and mentioned in the ECHA guidance as the in vitro test to be performed as part of weight of evidence.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™

Version / remarks:

March 2018

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The KeratinoSens™ assay is recommended in international guidelines (e.g. OECD 442D) for substitution of animal testing and mentioned in the ECHA guidance as the in vitro test to be performed as part of weight of evidence.

Specific details on test material used for the study:

The test item was weighed under nitrogen conditions (Atmos bag) with oxygen concentrations of 0.3% and 0.4% in the first and second experiment, respectively.

Details on the study design:

Based on a solubility test, a concentration of 200 mM in Ethanol (EtOH) was selected as highest concentration for the main assay (highest dose required in the current guideline).

In the main experiments the test item was dissolved in EtOH at 800 mM (colourless). From this stock 11 spike solutions in EtOH were prepared (2-fold dilution series in experiment 1 and 1.25-fold dilution series in experiment 2). The stock and spike solution were diluted 100-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM and 2000, 1600, 1280, 819, 655, 524, 419, 356, 268, 215 and 172 μM in experiment 1 and 2, respectively (final concentration EtOH of 0.25%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution.

Test item concentrations were used within 0.5 hours after preparation.

CONTROL ITEMS
- Positive control: ethylene dimethacrylate glycol (tested in triplicate)
Amount used: 0.78 to 25 mM in DMSO, diluted so that the final concentration ranged from 7.8 to 250 μM (final concentration DMSO of 1%)
- Negative control: eighteen wells per plate of the solvent control 0.25% EtOH and 1% DSMO were tested
- Blank control: on each plate three bank wells were tested (no cells and no treatment)

TEST DESIGN
- Test system: The KeratinoSens™ cell line, having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used. Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock.
- Plating of cells: For testing, cells were 80-90% confluent. The passage number used was P+14 in experiment 1 and P+7 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. The treated plates were sealed with foil to prevent evaporation and then incubated for about 48 hours ± 1 at 37±1.0°C in the presence of 5% CO2. In total 2 valid experiments were performed.
- Luciferase activity measurement: Plates with 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 KeratinoSens 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 - 4 hours at 37°C in the presence of 5% CO2. Cells were lysed and subsequently the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

DATA ANALYSIS: according to guideline

ACCEPTABILITY CRITERIA
The KeratinoSens™ 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 statistically significant 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 within two standard deviations of the historical mean. 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) The average coefficient of variation of the luminescence reading for the vehicle (negative) control EtOH should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded. If the variability is higher, a maximum of three of the eighteen wells may be excluded based on the Dixon’s Q-test. If the variability is still higher, the results should be discarded.
d) The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded. If the variability is higher, a maximum of three of the eighteen wells may be excluded based on the Dixon’s Q-test. If the variability is still higher, the results should be discarded.


INTERPRETATION
A KeratinoSens™ 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 KeratinoSens™ prediction is considered negative.
1. The Imax is equal or higher than (≥) 1.5 fold and statistically significantly different as compared to the vehicle (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 ≥ 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 and which do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should be considered as inconclusive.

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.00 and the EC1.5 58 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.70 and the EC1.5 63 µM.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

2.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: EC1.5: 1250 μM

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: EC1.5: Could not be calculated

Other effects / acceptance of results:

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 within two standard deviations of the historical mean (58 μM and 63 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.00-fold and 2.70-fold in experiment 1 and 2, respectively).
- The average coefficient of variation of the luminescence reading for the vehicle (negative) control EtOH was below 20% (8.6% and 5.8% in experiment 1 and 2, respectively).
- The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (4.3% and 5.6% in experiment 1 and 2, respectively).


No precipitation or cytotoxicity (IC30 and IC50) was observed.

Interpretation of results:

other: Study cannot be used for classification independently, but in a WoE for the end point Skin Sensitisation.

Conclusions:

Methylic Adduct is classified in the Keratinosens™ assay as negative since negative results (<1.5-fold induction) were observed at test concentrations < 1000 μM.

Executive summary:

A KeratinoSens assay was performed with Methylic Adduct according to OECD 442D; EURL ECVAM Protocol n° 155; and GLP principles. In the main experiments the test item was dissolved in EtOH at 800 mM (colourless). From this stock this stock 11 spike solutions were made with the test item in a concentration range of 0.98 – 2000 μM (2 -fold dilution steps) and 172 – 2000 μM (1.25 -fold dilution steps) in experiment 1 and 2, respectively. The highest test concentration in the first experiment was the highest dose required in the current guideline. In the second experiment, a narrower dose-response analysis was performed using a lower dilution factor of 1.25-fold to investigate the induction at 2000 μM in experiment 1 in more detail. No precipitate was observed at any dose level tested. Two independent experiments were performed. All acceptability criteria were met.

The test item showed no toxicity (no IC30 and IC50 value) in both experiments. In the first experiment the test item showed an induction of the luciferase activity (EC1.5: 1250 μM), however this induction was observed at test concentrations > 1000 μM and were therefore considered not biologically relevant. In the second experiment no EC1.5 value could be calculated. The maximum luciferase activity induction (Imax) was 2.12-fold and 1.12-fold in experiment 1 and 2, respectively. Methylic Adduct is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed at test concentrations < 1000 μM.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The skin sensitisation potential of the substance was assessed using the Weight of Evidence approach recommended, consisting of In silico assessment and in vitro assays.

Based on the current data it is concluded that Methylic Adduct does not have skin sensitizing properties. Therefore, it does not need to be classified as skin sensitizer according to Regulation (EC) No 1272/2008 (CLP) and related amendments.