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Administrative data

Description of key information

DPRA (OECD TG 442C): Test substance shows a minimal chemical reactivity

LuSens (OECD TG 442D): Test substance does not have a keratinocyte activation potential

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Parameter:
other: mean peptide depletion
Remarks:
Test substance
Run / experiment:
Cysteine and Lysine combined
Value:
0.43
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: mean peptide depletion
Remarks:
positive control
Run / experiment:
Cysteine and Lysine combined
Value:
36.63
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Substance identity

Cyteine-Peptide

Lysine-Peptide

Mean of both depletions [%]

Mean depletion [%]

SD [%]

Mean depletion [%]

SD [%]

Test substance

0.53

0.53

0.33

0.42

0.43

PC

53.52

2.39

19.74

1.61

36.63

PC, positive control (EGDMA in ACN)

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:
1. Preparation of the cells
LuSens cells from the working cell bank were thawed and cultured using culture medium, under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) for at least passage ≥5 but not longer than 15 passages prior to testing. Before substance incubation, cells were seeded in 96-well microtiter plates (120 μL of 0.83 x 105 cells/mL cell suspensions), using culture medium 2 for incubation for 24 hours. Three independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested.
2. Test-substance application for MTT and luciferase assay
After cell adaption for 24 hours cell culture medium 2 was aspirated and replaced with 150 μL medium. Each preparation of the dilution plate was then applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1%). After test substance application the plates were sealed with semi-permeable plate sealers in
order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours. For the luciferase assay a white plate (luminescence compatible plate) was used. In addition, a clear plate was treated in parallel for the determination of cell viability.
3. Visual inspections
Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 hours in order to detect test-substance precipitates.
4. Luciferase assay
After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Subsequently 200 μL of Steady-Glo-preparation (= 100 μL Steady-Glo- Mix and 100 μL PBS (without Ca2+/Mg2+)) per well was added and cells were shaken on a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.
5. Cell viability assay MTT
Cell culture medium was aspirated from all wells. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Thereafter 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and medium) was added to each well of the 96-well microtiter plate and incubated for further 2 hours after sealing the plates in the incubator. For analysis, medium was aspirated and cells were lysed by adding 100 μL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectralphotometer.
Parameter:
other: fold induction at highest valid concentration
Remarks:
805 ug/ml, rel. viability 71.1%
Run / experiment:
experiment 1
Value:
1.24
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: fold induction at highest valid concentration
Remarks:
805 ug/ml, rel. viability 70%
Run / experiment:
experiment 2
Value:
0.85
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: fold induction at highest valid conc.
Remarks:
559 ug/ml, rel. viability 76.3%
Run / experiment:
experiment 4
Value:
0.91
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the data available and the the criteria for classification laid down in Regulation (EC) 1272/2008 (CLP), non-classification of the test substance is warranted.