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EC number: 247-810-2 | CAS number: 26566-95-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data

Repeated dose toxicity: other routes
Administrative data
- Endpoint:
- repeated dose toxicity: other route
- Remarks:
- in vitro mechanistic study
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- February 20, 2019 - March 1, 2019
- Reliability:
- 2 (reliable with restrictions)
Data source
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: In vitro mechanistic assay that is currently being validated by OECD
- GLP compliance:
- no
- Limit test:
- no
Test material
- Specific details on test material used for the study:
- Multiple alkyl ZDDP substances and the base oil they are manufactured in were tested.
Test animals
- Species:
- other: six validated GFP-based mouse embryonic stem (mES) reporter cell lines
Administration / exposure
- Route of administration:
- other: in vitro
- Vehicle:
- DMSO
- Details on study design:
- TEST PROTOCOL
The ToxTracker assay requires only standard cell culture facilities and a low-end flow cytometer. The
ToxTracker reporter cells are maintained by culturing them in gelatin-coated dishes in the presence
of irradiated primary mouse embryonic fibroblasts (MEFs) in mES cell culture medium. During ch
emical exposures and reporter analysis the ToxTracker cells are cultured in the absence of fibroblasts
in mES cell culture medium.
Cytotoxicity testing/dose range finding
For chemical testing, first a dose range finding was performed using wild-type mES cells (strain B
4418). Wild type mES cells are exposed to 20 different concentrations of the test substances, with a
maximum concentration of 1% (see Table 3). Cytotoxicity is estimated by cell count after 24 h expos
ure using a flow cytometer and is expressed as percentage of viable cells after 24 h exposure compar
ed to vehicle control exposed cells. From this dose range finding, 5 concentrations are selected.
ToxTracker
The six independent mES reporter cell lines are seeded in gelatin-coated 96-well cell culture plates
in 200 μl mES cell medium (50.000 cells per well). 24 h after seeding the cells in the 96-well plates,emicals is added to the cells. For each tested sample, five concentrations are tested in 2-fold dilu
tions. Induction of the GFP reporters was determined after 24 h exposure using a flow cytometer. Onl
y GFP expression in intact single cells was determined. Mean GFP fluorescence was measured and
used to calculate GFP reporter induction compared to a vehicle control treatment. Cytotoxicity was
estimated by cell count after 24 h exposure using a flow cytometer and was expressed as percentage
of intact cells after 24 h exposure compared to vehicle exposed controls. For cytotoxicity assessment
in the ToxTracker assay, the relative cell survival for the six different reporter cell lines was average
d. Metabolic activation was included in the ToxTracker assay by addition of S9 liver extract from ar
oclor1254- induced rats (Moltox). Cells are exposed to five concentrations of the test samples in the
presence of 0.25% S9 and required co-factors (RegenSysA+B, Moltox) for 24 h.
Positive reference treatments with cisplatin (DNA damage), diethyl maleate (oxidative stress), tunica
mycin (unfolded protein response) and aflatoxin B1 (metabolic activation of progenotoxins by S9) wer
e included in all experiments. Solvent concentration was the same in all wells and never exceeded 1%
for the base oils. In case auto-fluorescence of the test substances was observed in the dose range
finding, wild type mES cells were exposed to the test samples at the same concentrations as used
in the ToxTracker. The mean fluorescence caused by the compound was then subtracted from the
ToxTracker results of the respective compound.
This experiment was conducted as a non-GLP study, however general principles to conduct proper
scientifically correct in vitro experiments were adhered to, and in particular care was taken for proper
handling of test article (stock) solutions to prevent/minimise degradation of the test articles based o
n instructions/compound information from the sponsor. For all ToxTracker analyses, Toxys strictly fo
llows the Good Cell Culture Practice guidelines from the OECD.
TEST CRITERIA
The ToxTracker assay was considered to have a positive response when a compound induces at l
east a 2 fold increase in GFP expression in any of the reporters. Activation of the Bscl2-GFP or Rtkn-
GFP reporters indicate induction of DNA damage, Srxn1-GFP and Blvrb-GFP indicate induction of cell
ular oxidative stress and Ddit3-GFP activation is associated with the unfolded protein response. The
Btg2-GFP reporter is controlled by the p53 tumor suppressor and is activated by DNA damage but
can also be induced by oxidative stress, hypoxia, metabolic stress and apoptosis.
Examinations
- Observations and examinations performed and frequency:
- Statistics
DATA ANALYSIS
In order to allow comparison of induction levels of the ToxTracker reporter cell lines for large
number of compounds we developed Toxplot, a dedicated data analysis software package.
Toxplot imports raw GFP reporter data from the flow cytometer, calculates GFP induction levels
and cytotoxicity, performs statistical analysis of the data and hierarchical clustering of the
tested compounds, and visualises the data in a heatmap allowing convenient interpretation of
obtained test results. ToxPlot software uses agglomerative hierarchical clustering to visualize the
ToxTracker data. Agglomerative clustering uses the ‘bottom-up’ approach, which puts each o
bservation in its own cluster and pairs of clusters are merged as one moves up the hierarchy. To com
pare the induction of the six GFP reporters for a collection of compounds, each with different biolo
gical reactivities, dose-response relationships and kinetics, Toxplot calculates for each compound the
level of GFP induction for every individual reporter at a specified level of cytotoxicity (typically 10%,
25% and 50%). GFP induction levels are calculated by linear regression between two data points
around the specified cytotoxicity level. In case the specified level of cytotoxicity can not be reached at
the highest tested compound concentration, Toxplot displays the GFP induction level at this top con
centration. In the heatmap, Toxplot clearly marks the compounds that do not induce the selected leve
l of cytotoxicity. Because the cytotoxicity for a compound can vary between the ToxTracker cell lines,
calculations of the GFP induction levels of the individual reporters by Toxplot can slightly deviate from
the GFP induction and cytotoxicity figures.
Results and discussion
Results of examinations
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- All ZDDP dosing was limited by cytotoxicity
- Description (incidence):
- Fold Activation for Reporter Genes
EC# Genotoxicity Cellular Stress Oxidative Stress Unfolded Protein Response
230-257-6 < 2.0 < 2.0 2.6 2.7
270-608-0 < 2.0 < 2.0 2.6 < 2.0
283-392-8 < 2.0 < 2.0 3.7 3.9
272-238-5 < 2.0 < 2.0 2.6 2.2
288-917-4 < 2.0 < 2.0 2.6 2.8
218-679-9 < 2.0 < 2.0 < 2.0 4.1
247-810-2 < 2.0 < 2.0 3.9 3.0
273-527-9 < 2.0 < 2.0 6.8 3.8
224-235-5 < 2.0 < 2.0 2.2 4.9
249-109-7 < 2.0 < 2.0 4.1 5.8
Base Oil 1 < 2.0 < 2.0 < 2.0 < 2.0
Base Oil 2 < 2.0 < 2.0 < 2.0 < 2.0
Base Oil 3 < 2.0 < 2.0 < 2.0 < 2.0
Base Oil 4 < 2.0 < 2.0 2.2 2.5
Positive Controls
Cisplatin 6.9 4.2 3.2 < 2.0
Diethyl Maleate < 2.0 4.1 31.5 2.0
Tunicamycin < 2.0 < 2.0 < 2.0 9.0
Alfatoxin B1 4.9 3.3 2.2 < 2.0
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