α,α,α-trifluorotoluene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2018-12-05 to 2019-02-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

α,α,α-trifluorotoluene

EC Number:

202-635-0

EC Name:

α,α,α-trifluorotoluene

Cas Number:

98-08-8

Molecular formula:

C7H5F3

IUPAC Name:

α,α,α-trifluorotoluene

In chemico test system

Details on the study design:

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

- cysteine peptide with an amino acid sequence of Ac-RFAACAA, JPT Peptide Technologies GmbH; >95 %; Lot. No.: 111016HS_MHeW0318
- lysine peptide with an amino acid sequence of Ac-RFAAKAA, JPT Peptide Technologies GmbH; >95 %; Lot. No.: 120514HSDW_W0318

Controls used:
- Positive control: Cinnamic aldehyde 100 mM in acetonitrile
- Co-elution control: test item or positive control without cysteine or lysine peptide
- Reference controls: cysteine or lysine peptide in acetonitrile with and without test item

Test substance preparation:
- The test substance was prepared as a 100 mM preparation in acetonitrile.

Peptide stock solution preparation:
- 20.69 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (39.36 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- 18.97 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (35.13 mL) to reach a concentration of 0.667 mM.

Experimental procedure:
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). Three samples of the test substance in acetonitrile were incubated with each peptide for 24 ± 2 h at room temperature (25 ± 2.5 °C) in the dark. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

HPLC conditions:
HPLC/DAD: Agilent Infinity 1260 II with Chromeleon 7.2 SR5
Detection: 220 nm signal for quantitation, 258 nm signal used as indicator for co-elution
Analytical Column: Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 μ m, Agilent Art. Nr. 861753-902
Pre-Column: Phenomenex, AJO-4286, 4.0 x 2.0 mm.
Column Temperature: 30 °C
Sample Temperature: 20 - 25 °C
Run Time: 20 minutes

Gradient:
Time Flow %A %B
0 min 0.35 mL/min 90 10
10 min 0.35 mL/min 75 25
11 min 0.35 mL/min 10 90
13 min 0.35 mL/min 10 90
13.5 min 0.35 mL/min 90 10
20 min end run

Injection Volume: 4 μL
HPLC Mobile Phase A: 0.1 % ( v/v) trifluoroacetic acid in water
HPLC Mobile Phase B: 0.085 % ( v/v) trifluoroacetic acid in acetonitrile

Calculation and data evaluation:
The concentration of the cysteine and lysine peptide was determined in each sample form absorbance at A = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions. PPD = (1- (Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in Reference Control C)) * 100

Acceptance criteria:
The run meets the acceptance criteria if:
- the standard calibration curve has a r2 > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8 % and 100 % for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9 %,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2 % and 69.0 % for the lysine peptide and the maximum SD for the positive control replicat es is < 11.6 %,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0 %.
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9 % for the cysteine per cent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6 % for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Evaluation of results:
Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser in accordance with UN GHS "Category 1", if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as "0" when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM. By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) the threshold of 6.38 % average peptide depletion was used to support the discrimination between skin sensitisers and nonsensitisers. By using the prediction model 2 (cysteine 1:10 prediction model) the threshold of 13.89 % peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.

Results and discussion

Positive control results:

The positive control caused depletion of both peptides comparable to historical data.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

mean

Parameter:

other: combined peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

mean

Parameter:

other: cysteine peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Run / experiment:

mean

Parameter:

other: lysine peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No precipitation or turbidity was observed for the samples of the test item. No phase separation with the cysteine peptide solution was observed. Slight phase separation with the lysine peptide solution was observed for all the samples of the test item. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.
- Co-elution: No relevant co-elution of the test item with any of the peptide peaks was observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. The test item might be considered as "non-sensitiser".

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. In the present study, performed according to OECD 442C, the test item was given into acetonitrile, based on the results of the pre-experiments. The test item was completely soluble and the resulting solution was used for further testing. Based on a molecular weight of 146.11 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control including the co-elution control. Slight phase separation was also observed for all the samples of the test item. Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

No co-elution of test item with the peptide peaks was observed. Slight phase separation in the lysine experiment was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control.

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 13.89 % (0.00 %). Based on the prediction model 2 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86 %.

Under the given conditions the test item showed minimal reactivity towards the cysteine peptide. The test item might be considered as “non-sensitiser”. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.