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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:
key study
Study period:
July - December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

In chemico test system

Details on study design:
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine-or lysine-containing peptide following 24 hours incubation with the test chemical at 25ºC. The synthetic peptides contain phenylalanine to aid in the detection. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteineand lysine peptide percent depletion values are then calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Used Chemicals:
Synthetic peptide containing Cysteine: Batch 1556171, Purity 95% (by HPLC), Ac-RFAACAA-OH
Synthetic peptide containing Lysine: Batch 1556172, Purtiy 94% (by HPLC), Ac-RFAAKAA-OH
Cinnamic Aldehyde (Positive control): Batch MKBR2427V, Purity 98.7%

Apparatus:
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Balances fitted with printers: Capability of weighing to 5 decimal places
General laboratory apparatus and glassware.

Preparation of Peptide Stock Solutions:
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).
Preparation of Peptide Calibration Standards:
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.
Preparation of Stability Controls and Precision Controls:
Stability controls (Reference Control B) and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile.
Preparation of Positive Control Stock Solution and Test Item Stock Solution:
100 mM stock solutions in acetonitrile of the positive control chemical (Cinnamic Aldehyde) and the test item were prepared.
Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls:
Acetonitrile solutions of the test item and the positive control were diluted with the Cysteine peptide to prepare solutions containing 0.5 mM Cysteine and 5 mM of either the test item or the positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.
Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls:
Acetonitrile solutions of the test item and the positive control were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either test item or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation:
The appearance of the test item and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Results and discussion

Positive control results:
Positive control values for cystein: 71,3 %, SD 0.25 %
Positive control values for lysine: 44.6 %, SD 0.87 %
The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be <14.9% for the percent cysteine depletion and <11.6% for the percent lysine depletion. The critera are met.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: cystein depletion (%)
Run / experiment:
1
Value:
> -2.26 - < -1.4
Vehicle controls valid:
not examined
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Parameter:
other: Mean lysine depletion (%)
Run / experiment:
1
Value:
> -0.08 - < 0.456
Vehicle controls valid:
not examined
Negative controls valid:
not examined
Positive controls valid:
yes
Other effects / acceptance of results:
All analytical acceptance criteria were met:
Acceptance criteria for Cysteine and Lysine (Standard Linearity): r²>0.99, achieved results: r²>0.999
Acceptance criteria for Cysteine and Lysine respectively (Reference controls): 0.45-0.55 mM (CV <14.9%) and 0.45-0.55 mM (CV <11.6%), achieved results: B: 0.499 mM (CV 0.75%, n=6) and B: 0.504 mM (CV 0.32%, n=6)
Acceptance criteria for Cysteine and Lysine (Test item): SD<14.9% and SD<11.6%, achieved results: SD 0.43% (n=3) and SD 0.30% (n=3)

Reactivity is classed as no to minimal, the DPRA prediction is negative and the test item is therefore a potential non skin sensitizer. A partial co-elution peak was observed in the Cysteine assay, however this is considered to be of minimal impact as the cysteine peak was consistently integrated in all three sample chromatograms. No co-elution peaks occurred in the Lysine assay.

Any other information on results incl. tables

Table 5: Individual Achieved Depletion Values of Cysteine Peptide Depletion

Sample

Peak area (μV.sec)

Peptide concentration(μg/mL) 1

Peptide Depletion (%) 2

Mean Depletion (%)

SD (%)

Positive control

253903

107.35

71.4

71.3

0.25

257128

108.71

71.0

252885

106.91

71.5

Test item

902625

382.01

-1.72

-0.179

0.43

899767

380.80

-1.40

907371

384.02

-2.26

 

SD Standard Deviation

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

2 Calculated against a mean Reference Control area of 887340 μV.sec (n=6)

 

 

Table 6: Individual Achieved Depletion Values of Lysine Peptide Depletion

Sample

Peak area (μV.sec)

Peptide concentration (μg/mL) 1

Peptide Depletion (%) 2

Mean Depletion (%)

SD (%)

Positive control

428621

212.34

45.6

44.6*

0.87

439007

217.50

44.3

441606

218.79

43.9

Test item

784117

389.07

0.456

0.112

0.30

788402

391.20

-0.0880

787965

390.98

-0.0320

SD Standard Deviation

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

2 Calculated against a mean Reference Control area of 787710 μV.sec (n=6)

* Individual and mean results are all outside of the historical data achieved parameters however all results are within acceptance criteria for the assay.

 

Applicant's summary and conclusion

Interpretation of results:
other: no skin sensitizing potential based on the key event "protein reactivity"
Conclusions:
In the Direct Peptide Reactivity Assay (DPRA) according to OECD 422C the test item showed no to minimal reactivity and is therefore predicted to be a non-skin sensitizer.
Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of the test item. All analytical acceptance criteria for each peptide run were met. Solutions of the test item were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. There was no reactivity (no depletion) in the presence of either peptide which places the test item in the reactivity class of “no or minimal reactivity” and therefore it is predicted by DPRA to be a non-skin sensitizer.