Registration Dossier

Administrative data

Description of key information

Based on the results of thein vitro skin sensitisation study, Alkaterge E is considered to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
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
Specific details on test material used for the study:
Test Material Name: Alkaterge E
CAS Number: 68 140-98-7
Appearance: Liquid, brown, viscous
Molecular weight: 365.60 g/mol
Details on study design:
Cell Thawing Procedure
A cryovial of the cryopreserved KeratinoSens cells was thawed in a water bath at approximately 37°C (in a beaker containing 70% ethanol) for approximately a minute. Once thawed, the cryovial was immediately decontaminated with 70% ethanol and placed in a laminar flow hood. The cells were added to a sterile conical tube and diluted by slowly adding 9 mL pre-warmed Assay Medium. The cells were then centrifuged at approximately 200 x g for 5 minutes at room temperature. The supernatant was aspirated, the pellet was resuspended in approximately 15 mL of fresh pre-warmed Assay Medium, and then transferred into a T75 tissue culture flask. The flask was then incubated at 37 ± 1°C, 90 ± 10% humidity, and 5.0 ± 1% CO2 in air (standard culture conditions), until the KeratinoSens cells reached approximately 60% to 90% confluence.

Routine Culturing of the KeratinoSens Cells
When the cultures reached approximately 60 to 90% confluence, they were removed from the flask by trypsinization. The medium was aspirated and the cell sheet rinsed twice with approximately 10 mL of CMF-DPBS containing 0.05% EDTA. One mL of trypsinl/EDTA was added to cover the cell sheet. The flask was then placed into the incubator and incubated at standard culture conditions for 6-8 minutes, or until the cells became dislodged. When more than 50% of the cells became dislodged, the flask was rapped sharply against the palm of the hand. Then approximately 7 mL of Maintenance Medium was added to each 75 cm2 flask to obtain a single cell suspension and cells passaged at appropriate densities. The KeratinoSens cells were routinely passaged every 2-4 days.

Subculture of KeratinoSens Cells into 96-Well Plates
The KeratinoSens cells were subcultured into transparent Costar 96-well plates or white-walled Perkin Elmer plates when the flasks were approximateLy 60 to 90% confluent. The flasks were rinsed and trypsinized as previously described. The cells were resuspended in 5 mL of Assay Medium per flask. The concentration of cells in suspension was determined using a Coulter Counter. A cell suspension of 1.0 x l0 cells/mL in Assay Medium was prepared. One hundred [tL of the cell suspension was added to all but one well designated as a blank well. The stock cell suspension was mixed often to ensure a uniform distribution of cells into each well.

MTT Direct Reduction Test
The ability of Alkaterge E to directly reduce MTT was assessed at the same time of test article treatment in the definitive assays. A 1.0 mg/mL MTT solution was prepared by dissolving a 10 mg/mL stock solution of MTT into warm MTT Addition Medium. Approximately 100 tL of the 100X test article concentration in DM50 was added to 1 mL of the M’T]’ solution and then incubated in the dark at 37°C for one to three hours. One hundred µL of a negative control (e.g. DMSO) was tested concurrently. If the MTT solution color turned blue/purple, the test articles was presumed to have reduced the MiT. The test article did not directly reduce MTT.

Testing Concentrations
Alkaterge E has a molecular weight of 365.6 glmol. The test article was tested at 100x stock dilution prepared to a top concentration of 200,000 µM. The final 1x tested concentrations were 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95 and 0.977 µM.

Definitive Assays
Alkaterge E was tested in three definitive assays. Each definitive assay included a set of 4 plates (3 for gene induction, I for cytotoxicity assessment). Each plate contained up to seven test articles with a range of 12 dosing concentrations for each test article. Each plate also included 5 wells designated for the positive control (tested over a range of 5 dosing concentrations), 6 wells designated as the DMSO negative (solvent) control, and I vell which was left blank.
Each definitive assay was performed independently: I) The plates for each definitive assay were seeded using separate flasks of cells 2) A separate IOOX Assay Master plate was prepared for each definitive assay 3) Three Separate 4x Mmaster plates were prepared and used to dose each set (4 Total Plates) of definite assay plates; and 4) Separate batches of MTT were prepared for each definitive assay.

After approximately 24 hours of incubation, at 37 ± 1°C, 5% C02, the Assay Medium was removed from the cells. The plates were decanted and gently blotted on sterile paper towels. One hundred and fifty microliters of fresh pre-warmed 1% DMEM were added to all wells, including the blank. The plates were returned to the incubator until the dosing was initiated. For each test article, twelve decreasing doses were selected for the assay (test articles were
diluted up to a final concentration of 200,000 µM in DMSO). For the positive control, 5 decreasing doses were prepared. For each experiment, the positive control (5 doses), and the negative (solvent) control, a l00x DMSO master plate was made, followed by a 4x Master Plate. When added to the 150 µL of 1% DMEM already in each well, the addition of the 50 p.L4X dose brought the final dose on the plates to lx.M in DMSO). For the positive control, 5 decreasing
doses were prepared. For each experiment, the positive control (5 doses), and the negative (solvent) control, a l00x DMSO master plate was made, followed by a 4x Master Plate. When added to the 150 µL of 1% DMEM already in each well, the addition of the 50 p.L4X dose brought the final dose on the plates to lx.
Treatment Termination & Luciferase Induction Determination
After 48 ±1 hours of exposure, each white-wailed culture plate was removed from the incubator and allowed to equilibrate to room temperature for at least 30 minutes. Once at room temperature, the treatment medium was decanted from each plate. The cultures were rinsed with 250 iL of CMF-DPBS (room temperature), the CMF-DPBS rinsate was decanted from the wells, and the plates were gently blotted onto paper towels. Fifty microliters of CMF-DPBS was added to each well followed by fifty microliters of ONEGloTM Reagent. The plates remained at room temperature in the dark for at least 5 minutes before being read by the luminometer. The plates were read within 30 minutes of addition of the ONE-Glo(TM) Reagent. The luminescense determination of each plate was performed by a Orion Berthold luminometer (Berthold Detection Systems, Pforzheim, Germany) initiated from an IBMPC hosting the Windows-based Simplicitytm’ software. The light intensity in each well was measured at 565 nm in the form of relative light units (RLU5).

Treatment Termination: Cytotoxicity Using the MTT Endpoint
A 0.59 mg/mL Mfl solution was prepared in 1% DMEM and used within 2 hours. After 48 ±1 hours, the clear 96-well plates designated for the MTT endpoint were decanted and gently hlotted on paper towels. No rinsing was performed. Two hundred [IL of 1% DMEM containing 0.59 mg/mL MTT was added to each well. The plate was incubated with a plate seal at 37 ± 1°C, 5% CO2 for approximately 4 hrs. After approximately 4 hours, the MTT solution was decanted, the plate was blotted, and 200 jiL of 10% SLS was added to each well. The plate was covered with a plate seal and incubated at 37 ± 1°C, 5% C02 overnight.
After the overnight incubation, each plate was placed on a plate shaker and shaken for at least 20 minutes at room temperature. The absorbance at 570 nm (OD570) of each well was measured with a Molecular Devices Vmax plate reader.
Positive control results:
The positive control, cinnamic aldehdye, showed the expected positive response in this assay.
Key result
Parameter:
other: mean EC1.5 (µM)
Run / experiment:
mean of assays
Value:
8.37
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: IC50 (µM)
Run / experiment:
mean of 3 assays
Value:
51.08
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: mean Imax
Run / experiment:
mean of 3 assays
Value:
3.59
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: mean CImax (µM)
Run / experiment:
mean of 3 assays
Value:
31.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
According to the current prediction model, the test article Alkaterge E, was predicted to be a sensitiser.
Executive summary:

The Induction of Antioxidant-Response-Element Dependent Gene Activity in the Keratinocyte ARE- Reporter Cell Line KeratinoSens assay was used to assess the skin sensitization potential of the Alkaterge E. The skin sensitization potential of the test article was evaluated using the protocol that is consistent with the OECD Test Guideline 442D “hi Vitro Skin Sensitisation: ARE-Nr12 Luciferase Test Method”. A test substance, was predicted to have sensitization potential if: l)The ECI.5 value fell below 1000 pM in at least 2 of 3 repetitions; 2) At the lowest concentration with a gene induction above 1.5, cellular viability was greater than 70%; and 3) There was an apparent overall dose response which was similar between the three definitive assays. According to the

current prediction model, the test article Alkaterge E, was predicted to be a sensitiser.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

Based on the results of the in vitro skin sensitisation study, Alkaterge E is self-classified as a skin sensitiser (category 1b), according to the criteria for classification of the CLP/GHS Regulation (EC) 1272/2008.