Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-11-16 to 2017-12-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Qualifier:
according to guideline
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-Fluor-4'-(trans-4-propylcyclohexyl)-1,1'-biphenyl-4-boronic acid
EC Number:
620-455-4
Cas Number:
524709-74-8
Molecular formula:
C21 H26 B F O2
IUPAC Name:
3-Fluor-4'-(trans-4-propylcyclohexyl)-1,1'-biphenyl-4-boronic acid
Test material form:
solid

In chemico test system

Details of test system:
cysteine peptide, (Ac-RFAACAA-COOH)
lysine peptide (Ac-RFAAKAACOOH)
Details on the study design:
PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
20.32 mg cysteine peptide were pre-weighed in a vial and dissolved in a defined volume (39.475 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
22.01 mg lysine peptide were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (41.50 mL) to reach a concentration of 0.667 mM.
- Preparation of the test chemical solutions: The test item was pre-weighed into a glass vial and was dissolved in DMF. A stock solution with a concentration of 100 mM was prepared.
- Preparation of the positive controls, reference controls and co-elution controls:
Positive control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Co-elution control: Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide.
Reference control A was prepared using acetonitrile.
Reference control B was prepared using acetonitrile.
Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item.

INCUBATION
- Incubation conditions: The reaction solutions were left in the dark at 25 +/- 2.5 °C for 24 +/- 2 h.
- Precipitation noted:
For the 100 mM solution of the test item precipitation was observed when diluted with the cysteine peptide solution. Precipitation was observed for the samples of the test item (including the co-elution control of the test item).
For the 100 mM solution of the test item precipitation was observed when diluted with the lysine peptide solution. Phase separation was observed for the samples of the positive control (including the co-elution control of the positive control). Precipitation was also observed for the samples of the test item (including the co-elution control of the test item).

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: Peptide standards were prepared in a solution of 20% acetonitrile : 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer). A serial dilution of the peptide stock solution (0.667 mM) using the respective dilution buffer) was performed, resulting in 7 calibration solutions (0.534 mM, 0.267 mM, 0.134 mM, 0.067 mM, 0.033 mM, 0.017 mM, 0.000 mM)

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm
Vehicle / solvent:
other: DMF
Positive control:
cinnamic aldehyde

Results and discussion

Positive control results:
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 63.50%.

In vitro / in chemico

Resultsopen allclose all
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
cysteine depletion
Value:
0.21 %
At concentration:
100 mM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
lysine depletion
Value:
0 %
At concentration:
100 mM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Outcome of the prediction model:
no or minimal reactivity [in chemico]
Other effects / acceptance of results:
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
replicates 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
percent 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.

Both peptide runs and the test item results met the acceptance criteria of the test.

Any other information on results incl. tables

Cysteine and Lysine Values of the Calibration Curve

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD1

5221.6792

0.5340

4311.3857

0.5340

STD2

2648.5273

0.2670

2143.2170

0.2670

STD3

1327.5009

0.1335

1043.3549

0.1335

STD4

670.7400

0.0667

519.3511

0.0667

STD5

334.8562

0.0334

259.5146

0.0334

STD6

166.3505

0.0167

130.4601

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1609.4324

0.1633

68.11

68.17

0.05

0.08

1604.4860

0.1628

68.21

1605.4248

0.1629

68.19

Test Item

4991.4937

0.5090

0.00

0.21

0.33

157.69

4966.8535

0.5064

0.04

4939.1538

0.5036

0.60

Depletion of the Lysine Peptide

Lysine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1589.2585

0.1982

59.95

58.82

0.99

1.68

1662.4030

0.2073

58.11

1650.5492

0.2058

58.41

Test Item*

4080.3315

0.5063

0.00

0.00

0.00

n/a

4120.3213

0.5113

0.00

4055.7935

0.5033

0.00

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model 1

Mean Cysteine andLysine PPD

Reactivity Class

DPRA Prediction²

0.00% PPD 6.38%

 No or Minimal Reactivity

Negative

6.38% < PPD 22.62%

Low Reactivity

Positive

22.62% < PPD 42.47%

Moderate Reactivity

42.47% < PPD 100%

High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD

ReactivityClass

DPRA Predictio

0.00% PPD 13.89%

No or Minimal Reactivity

Negative

13.89% < PPD 23.09%

Low Reactivity

Positive

23.09% < PPD 98.24%

Moderate Reactivity

98.24% < PPD 100%

High Reactivity

Categorization of the Test Item

Prediction Model

Prediction Model 1
(Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)

Prediction Model 2
(Cysteine Peptide / Test Item Ratio: 1:10)

Test Substance

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Test Item

-

-

-

0.21

Minimal Reactivity

no sensitiser

Positive Control

63.50

High Reactivity

sensitiser

68.17

Moderate Reactivity

sensitiser

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. Due to the observed precipitation, the prediction model does not apply and a prediction cannot be made.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.
Executive summary:

In the present study the test material, the test item, was dissolved in DMF, based on the results of the pre-experiments.

Based on a molecular weight of 340 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 solution of the test item 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. Precipitation was observed for the samples of the test item (including the co-elution control of the test item). Samples were centrifuged at 400g for 5 minutes prior to the HPLC analysis.

For the 100 mM solution of the test item 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 of the positive control). Precipitation was also observed for the samples of the test item (including the co-elution control of the test item). Samples of the test item were centrifuged at 400g for 5 minutes 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 insignificant.

Co-elution of the test item with the lysine peptide peak was observed. Therefore 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 C (RC CDMF). However, precipitation was observed in the cysteine experiment as well. Since it cannot be determined if the precipitate resulted from the test item or the peptide, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

The 100 mM stock solution of the test item showed minimal reactivity towards the cysteine peptide. The mean depletion of cysteine peptide was  6.38% (0.21%).

According to the evaluation criteria in the guideline, if a precipitation or phase separation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed precipitation in the cysteine experiment no prediction can be made.

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 63.50%.