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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

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 2015
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted February 2015
Qualifier:
according to
Guideline:
other: OECD 442E (h-CLAT)
Version / remarks:
adopted July 2016
Qualifier:
according to
Guideline:
other: Commission Regulation (EU) 2017/735, B.59 (DPRA)
Version / remarks:
adopted February 2017
Qualifier:
according to
Guideline:
other: Commission Regulation (EU) 2017/735, B.60 (LuSens)
Version / remarks:
adopted February 2017
GLP compliance:
yes (incl. certificate)
Type of study:
other: Combined evaluation of direct peptide binding and activation of keratinocytes and dendritic cells

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: 99.324%
Batch: K3331 Sumpf
Appearance: liquid/ colorless, clear
Storage: ambient under nitrogen

In vitro test system

Details on study design:
All: no analysis of the test substance in the vehicle was performed, since it was applied shortly after preparation.

DPRA:
Synthetic peptides: Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol), Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
concentrations: 5% (50.3mg/ml) and undiluted (99.3% pure) - a gravimetric method was used, since no molecular weight could be assigned to the complex mixture of hydrocarbons (UVCB)
ratio for incubation:
5% dilution: 1:7 and 1:32 (cysteine and lysine containing peptides), based on absolute mass - tested as emulsion
undiluted: 1:10 and 1:50 (cysteine and lysine containing peptides) - tested as emulsion
vehicle controls (propanol) and all concentrations performed as triplicate
positive control: EGDMA (CAS 97-90-5)
co-elution control performed to detect possible interferences of the test substance with the peptides


LuSens (Keratinocyte activation)
cell line: Human transgenic keratinocyte cell line derived from HaCaT cells
cell densitiy: 0.83 x10e5 /ml
incubation time: 48h at 37°C
concentrations based on MTT assay:
first experiment
4.5, 5.4, 6.5, 7.8µg/mL
fourth experiment (experiments 2 + 3 did not meet acceptance criteria)
1.1, 1.3, 1.5, 1.8, 2.2, 2.6, 3.1, 3.8, 4.5, 5.4, 6.5µg/mL
next higher concentration caused a reduction in viability of more than 30%
dilutions were prepared by stirring
positive control: EGDMA, 18µg/ml (CAS 97-90-5)
negative control: DL-lactiv acid, 450µg/ml in 1% DMSO (CAS 50-21-5)
vehicle control 1% DMSO in culture medium
Replicates: triplicates


h-CLAT (activation of dendritic cells)
induction of CD86 and CD54
cell line: human monocytic leukemia cells THP-1
cell densitiy: 2x10e6/ml
incubation time: 24h at 37°C
cytotoxicity: determined by PI - DNA intercalation (goal: viability > 50%, due to a calcuation error for viability, too low concentrations were used in the first experiments)
concentrations:
first experiment: 40, 48, 58, 69, 83, 100, 120, 144µg/mL
second experiment: 83, 100, 120, 144, 173, 208, 249, 299µg/ml
third through fifith experiment: 120, 144, 173, 208, 249, 299, 359, 431µg/mL
Dilutions were prepared by stirring and ultra-sonication
positive control: DNCB, 4µg/ml (CAS 97-00-7)
negative control: DL-lactiv acid, 1000µg/ml (CAS 50-21-5)
Duplicates
vehicle control 0.2% DMSO in culture medium

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: Peptide depletion
Remarks:
(mean of both peptides)
Run / experiment:
5%
Value:
2.99
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable because of methodological limitations
Remarks:
substance tested as emulsion, so the test may be underpredictive
Parameter:
other: peptide depletion
Remarks:
mean of both peptides
Run / experiment:
undiluted
Value:
0.54
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable because of methodological limitations
Remarks:
potentially underpredictive, since substance tested as emulsion
Parameter:
other: EC 1.5 (µg/ml)
Remarks:
(LuSens)
Value:
1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: EC200 (µg/ml)
Remarks:
CD54 (h-CLAT)
Run / experiment:
1
Value:
132
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
(no increase for CD 86)
Parameter:
other: EC200 (µg/ml)
Remarks:
CD54 (h-CLAT)
Run / experiment:
5
Value:
50
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
(no increase for CD86)
Other effects / acceptance of results:
DPRA
no Co-elution of the substance with the peptide occurred

LuSens
no precipitation occurred

h-CLAT
no precipitation occurred

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
DPRA: negative, but potentially underpredictive, since substance tested as emulsion. Result thus rendered inconclusive
LuSens: positive (EC1.5 = 1.5µg/mL9
h-CLAT: positive (EC 200 >=50 <=132µg/mL) based on CD54 induction. No induction of CD86
Overalls interpretation: positive