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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:
04 - 08 Oct 2016
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:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
yes
Remarks:
No information on stability, precision and co-elution controls provided. No information on stability/solubility of test substance after preparation of peptide depletion samples and after 22 h incubation.
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

In chemico test system

Details on 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: 1556171
Molecular weight: 751.5 g/L
Purity: 95%
Expiry date: 5 years
- Synthetic lysine-containing peptide:
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Molecular weight: 776 g/L
Purity: 90 - 95%
Expiry date: 5 years

SOLVENT CONTROL AND ASSESSMENT OF TEST ITEM SOLUBILITY
- Solvent: acetonitrile
The solubility of the test substance in acetonitril was assessed at a concentration of 100 mM.

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Batch number: MKBR2427V
- Purity: > 95%
- Expiry date: Feb 2019
The positive control was prepared at a concentration of 100 mM in acetonitrile.

STABILITY AND PRECISION CONTROL
Stability and precision controls of both peptides were prepared at a concentration of 0.5 mM.

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
- Peptide to test substance ratios: Cysteine-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance/positive control); Lysine-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance/positive control)
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: at least 22 h prior to initiation of the analysis run

NUMBER OF REPLICATES
for each peptide in triplicates for treatment and control

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm with guard 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
% B: 10, 25, 90, 90, 10, 10
- Detector Wavelength: 220 nm
- Calibration standard concentrations of both peptides: 0.0167, 0.0334, 0.0667, 0.133, 0.267 and 0.534 mM
- Column temperature: 30 °C

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: % depletion of cystein-containing peptide
Remarks:
(mean value of 3 replicates)
Run / experiment:
≥ 22 h incubation
Value:
4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: % depletion of lysine-containing peptide
Remarks:
(mean value of 3 replicates)
Run / experiment:
≥ 22 h incubation
Value:
-0.29
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid
Other effects / acceptance of results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: No co-elution of the test substance occurred during the assay. The solubility of the test substance in acetonitrile at a nominal concentration of 100 mM was confirmed.

ACCEPTANCE OF RESULTS:
All analytical acceptance criteria for each peptide were met.

Any other information on results incl. tables

Table 5. Mean peptide depletion of cysteine-containing peptide.

 

 

Peak area (µV.sec)

Peptide concentration

(µg/mL)*

Peptide depletion (%)**

Mean depletion ± CV (%)

 

Positive control

251471

106.80

71.6

71.1 ± 1.57

257737

109.47

70.9

258935

109.98

70.7

 

Test substance

850838

362.55

3.82

4.00± 0.41

845272

360.17

4.45

851605

362.87

3.73

 

CV: Coefficient of Variation

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

**: Calculated against a mean reference control peak area of 884600μV.sec (n=6)

Table 6. Mean peptide depletion of lysine-containing peptide.

 

 

Peak area (µV.sec)

Peptide concentration

(µg/mL)*

Peptide depletion (%)**

Mean depletion ± CV (%)

 

Positive control

318704

158.32

59.5

58.8 ± 2.29

321321

159.62

59.2

332679

165.29

57.7

 

Test substance

789247

392.99

-0.261

-0.290 ± 0.18

790939

393.84

-0.476

788209

392.47

-0.129

CV: Coefficient of Variation

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

**: Calculated against a mean reference control peak area of 787190μV.sec (n=6)                            

 

 

Applicant's summary and conclusion

Interpretation of results:
other: DPRA prediction: negative (no or minimal reactivity) according to OECD 442C
Conclusions:
Under the conditions of the Direct Peptide Reactivity Assay the test substance showed no or minimal peptide reactivity.
Executive summary:

The protein reactivity of the test substance was investigated in a Direct Peptide Reactivity Assay according to OECD 442C and in compliance with GLP (2017). In this study the test substance showed no or minimal peptide reactivity