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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
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes (incl. QA statement)
Type of study:
other: LuSens Test
Justification for non-LLNA method:
The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens As-say). The assay differs in some points from the OECD guideline.

Test material

Constituent 1
Chemical structure
Reference substance name:
Diethyl ethylphosphonate
EC Number:
201-111-9
EC Name:
Diethyl ethylphosphonate
Cas Number:
78-38-6
Molecular formula:
C6H15O3P
IUPAC Name:
diethyl ethylphosphonate
Test material form:
liquid
Specific details on test material used for the study:
Designation in Test Facility: 17080101G
Date of Receipt: 31. Jul. 2017
Condition at Receipt: Room temperature
6.1.1 Specification
The following information concerning identity and composition of the test item was provid-ed by the sponsor.
Name Diethyl ethylphosphonate
Batch no. 170635
Appearance colorless clear liquid
Composition Diethyl ethylphosphonate
Purity 96.5 %
Homogeneity homogeneous
Expiry date 18. Jun. 2019
Storage Room Temperature (20 ± 5°C)
The following information was provided by the sponsor as well:
CAS No. 78-38-6
EINECS-No. 201-111-9
Molecular formula C6H15O3P
Molecular weight 166.16 g/mol
Vapour pressure not stated
Stability H2O: 96h; EtOH: 96h; acetone: 96h; CH3CN: 96h; DMSO: 96h
Solubility H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Production date 19. Jun. 2017

In vitro test system

Details on the study design:
6.3.1 Reasons for the Choice of the LuSens Cell Line
The LuSens cell line was specially designed for this test system by the BASF SE (Lud-wigshafen, Germany). It employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany) carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck).
6.3.2 Cell Cultures
For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitro-gen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
For the Cytotoxicity Range Finder Assay cells of passage 7 were used. For the main ex-periments cells of passage 9 were used. After thawing the cells were cultivated in DMEM (9 % FCS) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Results and discussion

In vitro / in chemico

Results
Parameter:
other: relative viability
Value:
70
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The average induction for the positive control should be ≥ 2.5 fold and it should have a rela-tive viability of at least 70 %.
The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70%.
The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.
At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.
All validity criteria were met. Therefore, the study is valid.

Applicant's summary and conclusion

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