Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-10-19 to 2017-10-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Qualifier:
according to guideline
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
GLP compliance:
yes (incl. QA statement)
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

Constituent 1
Chemical structure
Reference substance name:
Fluoren-9-one
EC Number:
207-630-7
EC Name:
Fluoren-9-one
Cas Number:
486-25-9
Molecular formula:
C13H8O
IUPAC Name:
9H-fluoren-9-one

In chemico test system

Details on the 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 66.16%.

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion [%]
Value:
0.29
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: lysine run
Parameter:
other: mean peptide depletion [%]
Value:
0
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

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD1

3309.9390

0.5340

4455.4287

0.5340

STD2

1616.4630

0.2670

2248.6604

0.2670

STD3

764.3784

0.1335

1103.8511

0.1335

STD4

380.1269

0.0667

555.1791

0.0667

STD5

195.6536

0.0334

278.7628

0.0334

STD6

99.5359

0.0167

141.0930

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Depletion of the Cysteine Peptide

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

809.6354

0.1341

74.93

75.35

0.40

0.53

794.7175

0.1317

75.39

783.8397

0.1299

75.72

Test Item

3335.1116

0.5412

0.00

0.29

0.50

173.21

3201.0125

0.5196

0.87

3236.5881

0.5253

0.00

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

1768.9691

0.2117

57.97

56.98

1.07

1.87

1804.7944

0.2160

57.12

1858.1759

0.2224

55.85

Test Item

4236.3945

0.5071

0.00

0.00

0.00

--

4228.3511

0.5061

0.00

4216.3423

0.5047

0.00

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% PPD 13.89%

No or Minimal Reactivity

Negative

13.89% < PPD 23.09%

Low Reactivity

Positive

23.09% < PPD 98.24%

Moderate Reactivity

98.24% < PPD 100%

High Reactivity

Categorization of the Test Item

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

0.14

Minimal Reactivity

no sensitiser

0.29

Minimal Reactivity

no sensitizer

Positive Control

66.16

High Reactivity

sensitizer

75.35

Moderate Reactivity

sensitizer

Applicant's summary and conclusion

Interpretation of results:
other: Study cannot be used as stand-alone for conclusion on classification
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item can be 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.
Executive summary:

In the present study the test material, 9 -Fluorenone, was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 180 g/mol 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.

For the 100 mM solutionof the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Slight precipitation was observed for the samples of the positive control (including co-elution control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM 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 were inspected for precipitation, turbidity or phase separation. Phase separation (small droplets on the bottom of the vial) was observed for the samples of the positive control (excluding the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria the depletion range of the positive control was fulfilled, the observed precipitations and phase separation were regarded as insignificant.

No co-elution of test item with the peptide peaks was observed. Sensitising 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 Cacetonitrile).

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% (0.14%). 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 66.16%.