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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:
2017-10-19 to 2018-01-18
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Qualifier:
according to
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Test material

Reference
Name:
Unnamed
Type:
Constituent

In chemico test system

Details on study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

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 63.81% (experiment 1) and 68.63% (experiment 2).

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run (experiment 1)
Value:
0.12
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run (experiment 1)
Value:
13.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run (experiment 2)
Value:
0.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run (experiment 2)
Value:
4.43
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
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 Experiment 1

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD1

4523.9644

0.5340

4169.8604

0.5340

STD2

2255.1311

0.2670

2102.4409

0.2670

STD3

1081.2599

0.1335

1026.2726

0.1335

STD4

538.3588

0.0667

515.4506

0.0667

STD5

264.6839

0.0334

262.0216

0.0334

STD6

131.3584

0.0167

131.2270

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Cysteine and Lysine Values of the Calibration Curve Experiment 2

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD1

17.870

0.5340

4663.5073

0.5340

STD2

9.012

0.2670

2361.9275

0.2670

STD3

4.518

0.1335

1192.6749

0.1335

STD4

2.252

0.0667

602.5394

0.0667

STD5

1.101

0.0334

302.3090

0.0334

STD6

0.530

0.0167

151.3044

0.0167

STD7

0.000

0.0000

0.0000

0.0000

Depletion of the Cysteine Peptide Experiment 1

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

1320.4471

0.1577

70.95

71.10

0.18

0.25

1315.4908

0.1571

71.06

1304.7782

0.1559

71.29

Test Item

4600.2661

0.5437

0.00

0.12

0.20

173.21

4583.2500

0.5417

0.00

4529.0562

0.5353

0.35

Depletion of the Cysteine Peptide Experiment 2

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

4.447

0.1325

74.33

74.15

0.18

0.25

4.477

0.1334

74.15

4.510

0.1344

73.96

Test Item

17.353

0.5175

0.00

0.30

0.52

173.21

17.167

0.5120

0.89

17.396

0.5188

0.00

Depletion of the Lysine Peptide Experiment 1

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

1690.0214

0.2164

56.37

56.51

0.47

0.83

1699.1774

0.2175

56.14

1664.2920

0.2131

57.04

Test Item

3573.1260

0.4572

7.76

13.20

5.57

42.20

3372.0542

0.4315

12.95

3141.8584

0.4020

18.89

Depletion of the Lysine Peptide Experiment 2

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

1539.7554

0.1748

64.31

63.12

1.08

1.71

1602.9802

0.1820

62.84

1630.8385

0.1852

62.20

Test Item

4162.2075

0.4752

3.52

4.43

0.86

19.40

4118.6553

0.4702

4.53

4088.5010

0.4667

5.23

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%PPD13.89%

No orMinimalReactivity

Negative

13.89% <PPD23.09%

LowReactivity

Positive

23.09% <PPD98.24%

ModerateReactivity

98.24% <PPD100%

High Reactivity

Categorization of the Test Item Experiment 1

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

6.66

Low Reactivity

sensitiser

0.12

Minimal Reactivity

no sensitiser

Positive Control

63.81

High Reactivity

sensitiser

71.10

Moderate Reactivity

sensitiser

Categorization of the Test Item Experiment 2

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

2.36

Minimal Reactivity

no sensitiser

0.30

Minimal Reactivity

no sensitiser

Positive Control

68.63

High Reactivity

sensitiser

74.15

Moderate Reactivity

sensitiser

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is considered as “non-sensitiser”.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.