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EC number: 298-577-9 | CAS number: 93819-94-4
10 of the ZDDPs demonstrated similar biological activity using six in vitro Toxys ToxTracker mechanistic
assays that measure four specific mechanisms of cellular toxicity. The ToxTracker assay is a panel of
six validated GFP-based mouse embryonic stem (mES) reporter cell lines that can be used to identify
the biological reactivity and potential carcinogenic properties of newly developed compounds in a single
test. The assay monitors activation of cellular signalling pathways using specific biomarkers for detection
of the biological reactivity of compounds. The activation of these biomarker genes is monitored using
generated green fluorescent (GFP) mES reporter cell lines. ToxTracker consists of a panel of six different
mES GFP reporter cell lines representing four distinct biological responses: general cellular stress, DNA
damage, oxidative stress and the unfolded protein response.
Based on an initial test with 64 compounds, the assay has been able to correctly predict the mode of
action for ~97% of the chemicals testing. The assay is currently undergoing OECD validation.
For the initial assay, 10 ZDDPs and 4 base oils were tested for cytotoxicity and mode of action. All
samples were tested with and without S9 activation to simulate metabolism. Positive reference
treatments with cisplatin (DNA damage), diethyl maleate (oxidative stress), tunicamycin (unfolded
protein response) and aflatoxin B1 (metabolic activation of progenotoxins by S9) were included in all
experiments. Solvent concentration was the same in all wells and never exceeded 1% for the base
oils. The ToxTracker assay was considered to have a positive response when a compound induces at
least a 2-fold increase in GFP expression in any of the reporters. Only GFP inductions at compound
concentrations that showed < 75% cytotoxicity are used for the ToxTracker analysis. To compare
the induction of the six GFP reporters for a collection of compounds, each with different biological
reactivities, dose-response relationships and kinetics, Toxys calculates for each compound the level of
GFP induction for every individual reporter at a specified level of cytotoxicity (typically 10%, 25% and
Based on the results of the assay, none of the four base oils exhibited any cytotoxicity, so all base oils
were tested up to 1%. All of the ZDDPs exhibited cytotoxicity, so the maximum concentration for the
ZDDPs was 0.002 – 0.06%. At least 5 serial dilutions were tested for each substance, and the assays
were repeated in triplicate.
The table below shows the highest fold activation of the reporter genes for each endpoint at compound
concentrations that cause 10 – 50% cytotoxicity. The 50% cytotoxicity cut-off was used as there was
significantly more variation in the results when the cytotoxicity was > 50%. As there generally were not
clear differences in reporter activation between when S9 was used and when it was not used, the worst
case value is used for each substance. Since DNA damage and oxidative stress have two reporter
genes that are used to measure activation of the pathway, the highest fold induction to give the worst
case response is used in the table.
Three of the four base oils did not activate any of reporter genes at all concentrations tested. The final
base oil slightly activated the pathways for oxidative stress and unfolded protein response without S9,
but the activation was only 2.2 – 2.5 fold. There was no activation of any reporter genes for the fourth
base oil when S9 was included in the assay. These results further support the conclusion that the base
oils themselves should not contribute to the ZDDP toxicity.
Of the 10 ZDDPs tested, none of the ZDDPs induced a 2-fold response in the reporter genes that
measured DNA damage either with or without S9. Therefore, all of the ZDDPs would be considered
negative for genotoxicity in this assay. In addition, none of the ZDDPs induced a 2-fold increase in GFP
expression for the cellular stress reporter genes either with or without S9.
Of the 10 ZDDPs tested, 9 / 10 activated the oxidative stress pathway. While there were some
differences with the fold induction varying from 2.2 – 6.8, all of the ZDDPs induced the pathways
significantly less than the positive control diethyl maleate (31.5-fold induction). In addition, 9 / 10 ZDDPs
activated the unfolded protein response pathway, causing a 2.2 – 5.8-fold induction. The positive control
caused a 9.0-fold induction.
Generally, activation of the different GFP reporters by the control compounds is fully compliant with
historical data thereby confirming the technical validity of the performed tests.
The key takeaway from these results is that the ZDDPs have very similar modes of action (oxidative
stress and unfolded protein response) when causing cellular toxicity. These assays further justify the
ZDDP category hypothesis. Further testing using this assay will be completed on all members of the
The full report is attached.
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