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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

Applicant's summary and conclusion

Conclusions:
The table above shows the highest fold activation of the reporter genes for each endpoint at compoun
d concentrations that cause 10 – 50% cytotoxicity. The 50% cytotoxicity cut-off was used as there w
as 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 re
porter 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 f
inal 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 positive control compounds is fully compl
iant 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
category.
Executive summary:

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

50%).

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 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

category.