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Toxicological information

Skin sensitisation

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
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.

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-dichloro-1,1,2-trifluoro-2-(trifluoromethoxy)ethane
EC Number:
219-094-1
EC Name:
1,2-dichloro-1,1,2-trifluoro-2-(trifluoromethoxy)ethane
Cas Number:
2356-53-8
Molecular formula:
C3Cl2F6O
IUPAC Name:
1,2-dichloro-1,1,2-trifluoro-2-(trifluoromethoxy)ethane
Test material form:
liquid
Details on test material:
Appearance: Colourless liquid
Storage conditions: At room temperature container flushed with nitrogen

In chemico test system

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.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
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.

Any other information on results incl. tables

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 (r2) 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.

            

Applicant's summary and conclusion

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.