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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:
03 Nov - 09 Nov 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
February 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Department of Health of the Government of the United Kingdom, Medicines & Healthcare products Regulatory Agency (MHRA), UK
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Pyridine-2-carboxylic acid
EC Number:
202-719-7
EC Name:
Pyridine-2-carboxylic acid
Cas Number:
98-98-6
Molecular formula:
C6H5NO2
IUPAC Name:
pyridine-2-carboxylic acid

In chemico test system

Details on the study design:
TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

TEST SYSTEM
- Supplier: AnaSpec
- Synthetic cysteine-containing peptide:
Alternative name: Ac-RFAACAA-OH
Batch number: 1658140
- Purity: 98%
- Synthetic lysine-containing peptide:
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Purity: 94%

SOLVENT CONTROL AND ASSESSMENT OF TEST ITEM SOLUBILITY
- Solvent: distilled water: acetonitrile, 1:1 (v/v)
- Batch number: 1673078, Fisher Chemical
- Purity: ≥ 99.9%
The test substance was soluble in distilled water:acetonitrile, 1:1 (v/v) after mixing 5 min ultrasonification at 100 mM.

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Batch number: MKCB9907, SAFC
- Purity: 99.1%
The positive control chemical (cinnamic aldehyde) was prepared at a concentration of 100 mM in acetonitrile.

STABILITY AND PRECISION CONTROL
Stability, precision and co-elution controls were prepared.

PEPTIDE STOCK SOLUTION PREPARATION
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM

INCUBATION CONDITIONS OF THE TEST SUBSTANCE WITH THE PEPTIDE SOLUTIONS
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: at least 22 h

NUMBER OF REPLICATES
triplicates for each peptide

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Water Alliance 2695 separation module and 2487 dual wavelength detector
- Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm with pre-column Phenomenex AJO4286
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in deionised water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 20, 21, 23, 23.5, 30
% A: 90, 75, 10, 10, 90, 90
% B: 10, 25, 90, 90, 10, 10
- Detector Wavelength: 220 nm
- Calibration standard concentrations of both peptides: 0, 0.0167, 0.0334, 0.0667, 0.133, 0.267 and 0.534 mM.
- Column temperature: 30 °C

Results and discussion

Positive control results:
Mean Cysteine Peptide Depletion of the Positive control ± standard deviation (%): 70.7 ± 0.51

Individual and mean concentrations of the positive control are outside of the range established during the validation study. There is however considered to be no impact on the data reporting as both each individual and the overall mean depletion values are within the stated acceptance criteria of 60.8% - 100% depletion.

Mean Lysine Peptide Depletion of the Positive control ± standard deviation (%): 58.0 ± 0.55

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: Cysteine containing peptide
Parameter:
other: % depletion of peptide
Remarks:
(mean value of 3 replicates)
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Lysine containing peptide
Parameter:
other: % depletion of peptide
Remarks:
(mean value of 3 replicates)
Value:
0.161
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Cysteine/Lysine containing peptide
Parameter:
other: % Overall mean depletion of peptide
Value:
0.081
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
- There was no co-elution peaks in either of the Lysine or Cysteine assays.
- The solubility of the test substance in acetonitrile at a nominal concentration of 100 mM was confirmed.

ACCEPTANCE OF RESULTS:
All analytical acceptance criteria were met.

Any other information on results incl. tables

 Table 3: The depletion of peptide in the presence of test substance

 

Mean peak area of reference control (µV.sec)

Mean peak area of peptide with test item (µV.sec)

Mean peptide depletion by test substance (%)

Cysteine

Control B: 847420 (n=6)

856730 (n=3)

-1.10

Lysine

Control B: 764530 (n=6)

763300 (n=3)

0.161

 

 

Table 4: Overall Achieved Depletion Values

Test item

Cysteine peptide depletion (%)

Lysine peptide depletion (%)

Overall mean depletion (%)

Reactivity class

DPRA prediction

Test substance

0.001*

0.161

0.0805

None to minimal

Negative

* overall negative result counts as a zero

 

Table 5: Cysteine Peptide Depletion

Sample

 

Peak area (μV.sec)

Peptide concentration1(μg/mL)

Peptide Depletion2(%)

Mean Depletion (%)

SD (%)

Positive control

248527

110.9533

70.7

70.7

0.51

244193

109.0133

71.2

252767

112.8533

70.2

Test Substance

857752

383.73

-1.22

- 1.10

0.13

856911

383.36

-1.12

855537

382.74

-0.958

SD Standard Deviation

1 Samples prepared at a concentration of 376 μg/mL (0.5 mM)

2 Calculated against a mean Reference Control B area of 847420 μV.sec (n=6)

3 Individual and mean concentrations of the positive control are outside of the range established during the validation study. There is however considered to be no impact on the data reporting as both each individual and the overall mean depletion values are within the stated acceptance criteria of 60.8% - 100% depletion.

 

Table 6: Lysine Peptide Depletion

Sample

 

Peak area (μV.sec)

Peptide concentration1(μg/mL)

Peptide Depletion2(%)

Mean Depletion (%)

SD (%)

Positive control

316216

161.34

58.6

58.0

0.54

323723

165.19

57.7

323072

164.86

57.7

Test Substance

761794

390.01

0.358

0.161

0.50

760485

389.33

0.529

767620

392.99

-0.404

SD Standard Deviation

1 Samples prepared at a concentration of 388 μg/mL (0.5 mM)

2 Calculated against a mean Reference Control B area of 764530 μV.sec (n=6)

 

 

Applicant's summary and conclusion

Interpretation of results:
other: DPRA prediction: no skin sensitising potential based on the key event “protein reactivity”.
Conclusions:
The mean depletion of cysteine and lysine was 0.0805% (mean% depletion ≤ 6.38%).
Under the conditions of the Direct Peptide Reactivity Assay the test substance showed no or minimal peptide reactivity. No skin sensitising potential based on the key event “protein reactivity” is predicted.
The present test alone does not suit for the non-classification or classification of the test substance as skin sensitiser; further testings should be considered.