Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-03-07 to 2016-04-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
adopted: February 04, 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154
Version / remarks:
January 12, 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
The Direct Peptide Reactivity Assay (DPRA) is accepted as a reliable and relevant replacement test for in vivo skin sensitisation testing.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Preparation of the Test Item
The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. The test item was pre-weighted into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.

In chemico test system

Details on study design:
The DPRA is supposed to address the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic model peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.
The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical.
This test may be used for the hazard identification of sensitising chemicals in accordance with UN GHS “Category 1”. It does not allow the classification of chemicals to the subcategories 1A and 1B as defined by UN GHS nor predict potency for safety assessment decisions. Therefore, all substances giving a positive result in the DPRA will be classified into UN GHS “Category 1”.
For detailed information on experimental procedure, materials, methods, controls, prediction model and acceptance criteria please see section “any other details on materials and methods incl. tables”.

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 62.90 %.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
Both Cysteine and Lysine
Value:
2.03
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: mean peptide depletion [%]
Run / experiment:
Cysteine
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Values were set to zero due to negative depletion
Parameter:
other: mean peptide depletion [%]
Run / experiment:
Lysine
Value:
4.07
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:
OTHER EFFECTS:
- Visible damage on test system: not reported
ACCEPTANCE OF RESULTS: all criteria were fulfilled. For details, please see Tables 10 and 11 in section “any other details on results incl. tables”.

Any other information on results incl. tables

Pre-Experiments

Solubility of the test item was determined prior to the main experiment. All test item solutions were freshly prepared immediately prior to use. The test item was not soluble in acetonitrile but completely soluble in water. No turbidity, precipitation and phase separation were observed for the test item solutions. All test item preparations of the main experiment were prepared using water.

Precipitation and Phase Separation

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution.

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 of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, positive control, RC A, RC B, RC C and for STD 1 and 2. Samples were not centrifuged prior to the HPLC analysis.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the any test item samples. Slight phase separation was observed for the positive control and the respective co-elution control.

After the HPLC run samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, positive control, RC A, RC B, RC C and for STD 1, 2 and 3.

After the HPLC run samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the any test item samples. Slight phase separation was observed for the positive control and the respective co-elution control.

Co-elution with the peptide peak

No co-elution of the test item with any of the peptide peaks was observed.

Results Calibration Curve

Table 6: 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

3495.8242

0.5340

3675.5410

0.5340

STD2

1805.4337

0.2670

1867.7395

0.2670

STD3

944.5201

0.1335

985.2802

0.1335

STD4

466.7887

0.0667

516.4465

0.0667

STD5

237.0339

0.0334

282.1445

0.0334

STD6

116.4341

0.0167

179.8809

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Based on these results, linear regression was performed and the following calibration curves were determined:

Cysteine Peptide Calibration Curve : y = 6546.17x + 26.29 ; R2= 0.9995

Lysine Peptide Calibration Curve : y = 6805.79x + 50.30 ; R2= 0.9996

 

Results of the Cysteine Peptide Depletion

Table 7: Depletion of the Cysteine Peptide. * Values were set to zero due to negative depletion.

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

906.7542

0.1345

71.54

72.27

0.90

0.01

892.3105

0.1323

71.99

851.7282

0.1261

73.27

Test Item

3217.3611

0.4875

0.00*

0.00

0.00

-

3224.2549

0.4885

0.00*

3206.4368

0.4858

0.00*

 

 Results of the Lysine Peptide Depletion

Table 8: 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

1608.7321

0.2290

53.57

53.53

0.22

0.00

1618.3588

0.2304

53.29

1603.4867

0.2282

53.72

Test Item

3266.0764

0.4725

4.10

4.07

0.10

0.02

3271.2881

0.4733

3.95

3264.7769

0.4723

4.14

Detailed results about the reference controls can be found in Table 15.

Categorization of the Test Item

Based on the results of the peptide depletion, categorization according to the prediction model was performed. In case that no co-elution was detected, the prediction model based on the combination of cysteine and lysine peptide should be used. Since no co-elution was observed the prediction model of cysteine and lysine was used.

Table 9:     Categorization of the Test Item

Predicition 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

2.03

Minimal reactivity

No sensitiser

0.00

Minimal reactivity

No sensitiser

Positive Control

62.90

High reactivity

sensitizer

72.27

Moderate reactivity

sensitizer

 

Acceptance Criteria

Table 10: Acceptance Criteria for Cysteine Peptide

Cysteine Peptide Run

Acceptance Criterion

Range

Value

pass/fail

coefficient of determination

R2> 0.99

0.9995

pass

mean peptide concentration of RC A

0.45 ≤ x ≤ 0.55 mM

0.4969

pass

mean peptide concentration of RC C (PC)

0.45 ≤ x ≤ 0.55 mM

0.4827

pass

mean peptide concentration of RC C (TI)

0.45 ≤ x ≤ 0.55 mM

0.4584

pass

CV of the peak area of RC B

< 15 %

2.38

pass

CV of the peak area of RC C (PC)

< 15 %

0.85

pass

CV of the peak area of RC C (TI)

< 15 %

9.07

pass

mean peptide depletion of the PC

60.8 % < x < 100 %

72.27

pass

SD of peptide depletion of the PC replicates

< 14.9 %

0.90

pass

SD of peptide depletion of the TI replicates

< 14.9 %

0.00

pass

Table 11: Acceptance Criteria for Lysine Peptide

Lysine Peptide Run

Acceptance Criterion

Range

Value

pass/fail

coefficient of determination

R² > 0.99

0.9996

pass

mean peptide concentration of RC A

0.45 ≤ x ≤ 0.55 mM

0.5067

pass

mean peptide concentration of RC C (PC)

0.45 ≤ x ≤ 0.55 mM

0.5017

pass

mean peptide concentration of RC C (TI)

0.45 ≤ x ≤ 0.55 mM

0.4930

pass

CV of the peak area of RC B

< 15 %

0.54

pass

CV of the peak area of RC C (PC)

< 15 %

0.61

pass

CV of the peak area of RC C (TI)

< 15 %

0.54

pass

mean peptide depletion of the PC

40.2 % < x < 69.0 %

53.53

pass

SD of peptide depletion of the PC replicates

< 11.6 %

0.22

pass

SD of peptide depletion of the TI replicates

< 11.6 %

0.00

pass

Table 12: Historical Data Cysteine Peptide

Cysteine Peptide

 

mean

SD

N

linearity of the calibration curve

0.9991

0.0006

8

mean peptide concentration of reference A [mM]

0.52

0.00

8

mean peptide concentration of reference C [mM]

0.50

0.00

10

CV of the peak area of control B [%]

2.10

0.34

8

CV of the peak area of control C [%]

1.60

0.85

10

mean peptide depletion of the PC [%]

74.67

2.32

8

SD of peptide depletion of the PC replicates [%]

0.84

0.72

8

SD of peptide depletion of the test items [%]

4.60

14.80

20

Table 13:   Historical Data Lysine Peptide

Lysine Peptide

 

mean

SD

N

linearity of the calibration curve

0.9998

0.0001

7

mean peptide concentration of reference A [mM]

0.49

0.02

7

mean peptide concentration of reference C [mM]

0.49

0.24

9

CV of the peak area of control B [%]

1.26

0.24

7

CV of the peak area of control C [%]

0.81

0.89

9

mean peptide depletion of the PC [%]

59.54

6.09

7

SD of peptide depletion of the PC replicates [%]

2.58

1.90

7

SD of peptide depletion of the test items [%]

1.02

1.08

21

Table 14:   Exemplary Analysis Sequence

Run 1

Run 2

Run 3

Run 4

Run 5

Run 6

Run 7

Run 8

Run 9

Run 10

Run 11

 

STD1

STD2

STD3

STD4

STD5

STD6

SDT7 (DB)

Reference Control A, replicate 1

Reference Control A, replicate 2

Reference Control A, replicate 3

Run 12

Run 13

Co-Elution Control Positive Control

Co-Elution Test Item 1

Run 14

Run 15

Run 16

Reference Control B, replicate 1

Reference Control B, replicate 2

Reference Control B, replicate 3

Run 17

Run 18

Run 19

Reference Control C, replicate 1

Positive Control, replicate 1

Test Item 1, replicate 1

Run 20

Run 21

Run 22

Reference Control C, replicate 2

Positive Control, replicate 2

Test Item 1, replicate 2

Run 23

Run 24

Run 25

Reference Control C, replicate 3

Positive Control, replicate 3

Test Item 1, replicate 3

Run 26

Run 27

Run 28

Reference Control B, replicate 4

Reference Control B, replicate 5

Reference Control B, replicate 6

Table 15: Results of the Reference Controls for the Cysteine Peptide

Cysteine Peptide Run

Sample

Peptide Peak Area

Peptide Concentration [mM]

PA

Mean

SD

CV [%]

Peptide Concentration

Mean

SD

CV [%]

Reference A 1

3295.67

3278.9512

26.0108

0.79

0.4994

0.4969

0.0040

0.80

Reference A 2

3292.20

0.4989

Reference A 3

3248.98

0.4923

Reference B 1

3211.87

3162.2388

74.5299

2.36

0.4866

0.4791

0.0114

2.38

Reference B 2

3253.24

0.4930

Reference B 3

3222.13

0.4882

Reference B 4

3097.92

0.4692

Reference B 5

3090.32

0.4681

Reference B 6

3097.95

0.4692

Reference C 1 (PC solvent)

3208.51

3186.06

26.9566

0.85

0.4861

0.4827

0.0041

0.85

Reference C 2 (PC solvent)

3193.52

0.4838

Reference C 3 (PC solvent)

3156.16

0.4781

Reference C 1 (TI solvent)

3205.63

3027.02

272.2805

8.99

0.4857

0.4584

0.0416

9.07

Reference C 2 (TI solvent)

2713.64

0.4105

Reference C 3 (TI solvent)

3161.80

0.4790

Table 16:   Results of the Reference Controls for the Lysine Peptide

Lysine Peptide Run

Sample

Peptide Peak Area

Peptide Concentration [mM]

PA

Mean

SD

CV [%]

Peptide Concentration

Mean

SD

CV [%]

Reference A 1

3484.17

3498.5079

12.6544

0.36

0.5046

0.5067

0.0019

0.37

Reference A 2

3508.10

0.5081

Reference A 3

3503.26

0.5074

Reference B 1

3472.14

3482.0649

18.3933

0.53

0.5028

0.5042

0.0027

0.54

Reference B 2

3506.05

0.5078

Reference B 3

3497.27

0.5065

Reference B 4

3473.01

0.5029

Reference B 5

3487.77

0.5051

Reference B 6

3456.15

0.5004

Reference C 1 (PC solvent)

3445.89

3465.01

20.6789

0.60

0.4989

0.5017

0.0030

0.61

Reference C 2 (PC solvent)

3462.18

0.5013

Reference C 3 (PC solvent)

3486.96

0.5050

Reference C 1 (TI solvent)

3426.42

3405.85

18.0704

0.53

0.4961

0.4930

0.0027

0.54

Reference C 2 (TI solvent)

3392.54

0.4911

Reference C 3 (TI solvent)

3398.60

0.4920

 

Applicant's summary and conclusion

Interpretation of results:
other: Expert judgement: no indication of sensitisation
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards the peptides. The test item can be considered as “non-sensitizer”. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and will be considered in the context of integrated approach.
Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential

of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP),

namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic peptides

containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine

peptide is used to categorize a substance in one of four reactivity classes to support discrimination

between skin sensitiser and non-sensitisers.

In the present study, the test item was dissolved in water and 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 analysis.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine and lysine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, RC A, RC B, RC C, positive control and for STD 1 and 2 of the cysteine run. No centrifugation was necessary to perform the HPLC analysis. Slight phase separation was observed for the positive control and the respective co-elution control of the lysine run.

After the HPLC run samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, RC A, RC B, RC C, positive control and for STD 1, 2 and 3 of the cysteine runs. Slight phase separation was observed for the positive control and the respective co-elution control of the lysine run.

Since the turbidity noted for the test item samples was also observed for reference controls, positive controls and standard solutions it can be considered that it is related to the peptide and that it is not a precipitation of the test substance. Additionally, the turbidity did not change during the HPLC analysis period. Since stability of the cysteine peptide in the used acetonitrile batch was demonstrated successfully, the reactivity of the positive control towards the cysteine peptide and peptide depletion were identified correctly and the validity of the cysteine run was acceptable the precipitation was considered as not relevant.

No co-elution of test item with the peptide peaks was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤6.38 % (2.03 %). Based on the prediction model 1 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 62.90 %.

The controls confirmed the validity of the study for both, the cysteine and lysine run. For the cysteine run the coefficient of determination for the calibration curve was >0.99 (0.9995). The mean peptide depletion of the cysteine peptide by the positive control was between 60.8 % and 100 % (72.27 %).

The mean peptide concentration of reference control A and reference control C (acetonitrile and water) was between >0.45 and <0.55 mM (RC A: 0.4969 mM, RC C (acetonitrile): 0.4827 mM, RC C (water): 0.4584 mM). The coefficient of variation (CV) of the peak area (PA) of reference control B and reference control C (acetonitrile and water) was <15 %. (RC B: 2.38 %, RC C (acetonitrile): 0.85 %, RC C (water): 9.07 %). The SD of the peptide depletion for the replicates of the positive control as well as for the tested test item samples was <14.9 % (PC: 0.90 %; test item 0.00 %).

For the lysine run the coefficient of determination for the calibration curve was >0.99 (0.9996). The mean peptide depletion of the lysine peptide by the positive control was between 40.2 % and 69.0 % (53.53 %). The mean peptide concentration of reference control A and reference control C (acetonitrile and water) was between >0.45 and < 0.55 mM (RC A 0.5067 mM, RC C (acetonitrile): 0.5017 mM, RC C water): 0.4930 mM). The coefficient of variation (CV) of the peak area (PA) of reference control B and reference control C (acetonitrile and water) was <15 %. (RC B: 0.54 %, RC C (acetonitrile: 0.61 %, RC C water): 0.54 %). The SD of the peptide depletion for the replicates of the positive control as well as for the tested test item samples was <11.6 % (PC: 0.22 %; test item: 0.10 %).

The test item can be considered as non-sensitiser. The data generated with this test will be considered in the context of an integrated approached such as IATA, combining this result with other complementary information.