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A DEREK assessment, DPRA assay and KeratinoSensTM assay were
performed. Based on these data ROC-118 was concluded to be a skin
sensitizer. To determine potency an LLNA study was performed.
DEREK NEXUS version 5.0.2 predicted the main constituent and both
impurities of ROC-118 to be sensitising to the skin (plausible).
4 Acceptability of the DPRA assay
Cysteine reactivity assay
Lysine reactivity assay
Results for SPCC
Results for SPCL
Correlation coefficient (r2) standard calibration curve
Mean peptide concentration RC-A samples (mM)
0.50 ± 0.05
0.485 ± 0.027
0.516 ± 0.020
Mean peptide concentration RC-C samples (mM)
0.513 ± 0.007
CV (%) for RC samples
B and C
Mean peptide depletion cinnamic aldehyde (%)
SD of peptide depletion cinnamic aldehyde (%)
SD of peptide depletion for Perfluoro methoxy dioxole (%)
RC = Reference Control; CV = Coefficient of Variation; SD =
Standard Deviation; NA = Not Applicable.
* For calculation of the RC-C mean and the CV, the RCcysC-3 value was
excluded (outlier due to injection error). As a result, no SD could be
calculated for the mean of the RCcysC.
5 SPCC and SPCL depletion and reactivity classification forPerfluoro
Mean of SPCC and SPCL depletion
Cysteine 1:10 / Lysine 1:50 prediction model
In an in chemico study, performed according to OECD guideline 442C
and GLP principles, the reactivity of ROC-118 towards model synthetic
peptides containing either cysteine (SPCC) or lysine (SPCL) was
determined to assign the test chemical to one of four reactivity classes
used to support the discrimination between skin sensitisers and non skin
Following incubation of the test substance with either SPCC or
SPCL, the relative peptide concentration was determined by
High-Performance Liquid Chromatography (HPLC) with gradient elution and
photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL
Percent Depletion Values were calculated and used in the prediction
Acetonitrile (ACN) was found to be an appropriate solvent to
dissolve the test substance, and was therefore used in this DPRA study.
Cinnamic aldehyde was used as a positive control.
The validation parameters, i.e. calibration curve, mean
concentration of Reference Control (RC) samples A and C, the CV for RC
samples B and C, the mean percent peptide depletion values for the
positive control with its standard deviation value and the standard
deviation value of the peptide depletion for the test substance, were
within the acceptability criteria for the DPRA assay. Therefore, the
study was considered to be valid.
No co-elution of the test item with SPPC or SPCL was observed.
the cysteine reactivity assay the test item showed 39.6% SPCC depletion,
and in the lysine reactivity assay 65.6% SPCL depletion. The mean of the
SPCC and SPCL depletion was 52.6%. As a result ROC-118 was classified in
the “high reactivity class” when using the Cysteine 1:10/Lysine 1:50
prediction model. Therefore, ROC-118 was considered to be positive in
A KeratinoSens(TM) assay was performed with ROC-118 according to OECD
442D and GLP. The test substance was dissolved in Dimethyl sulfoxide at
200 mM. From this stock 11 spike solutions in DMSO were prepared. The
stock and spike solutions were diluted 100-fold in the assay resulting
in test concentrations of 0.060 – 125 μM (2-fold dilution series). The
highest test concentration was considered to be the limit of solubility.
The test item precipitated at the highest dose level tested. Two
independent experiments were performed, which both passed the acceptance
The test item showed toxicity in the first experiment (IC30 value of 40
μM). A biologically relevant, dose-related induction of the luciferase
activity (EC1.5 values of 0.90 μM and 0.70 μM in experiment 1 and 2,
respectively) was measured in both experiments. The maximum luciferase
activity induction (Imax) was 68.36-fold and 59.21-fold in experiment 1
and 2 respectively. ROC-118 is classified as positive in the
KeratinoSens(TM) assay since positive results (>1.5-fold induction) were
observed at test concentrations <1000 μM with a cell viability of >70%
compared to the vehicle control.
DEREK NEXUS was performed for the main component and impurity 1.
DEREK NEXUS version 5.0.2 yielded two alerts for the main component of
ROC-118 for skin sensitization based on the presence of the phenyl ester
group and the alpha,beta-unsaturated ester group. The main component is
predicted to be sensitizing to the skin (plausible). DEREK NEXUS
predicts an EC3 of 8.1% (moderate sensitizer) for the phenyl ester group
and 79% (weak sensitizer) for the alpha,beta-unsaturated ester group
based on data on closest structurally-related substances. The mechanism
for the first alert is thought to occur through skin protein acylation
following nucleophilic attack of skin proteins at the carbonyl carbon of
the ester group; for the latter alert the Michael addition applies.
For impurity 1 the same alerts as for the main component were
presented with a predicted EC3 of 5.9% (moderate sensitizer) for the
phenyl ester group and 58% (weak sensitizer) for the
alpha,beta-unsaturated ester group. DEREK NEXUS predicted also skin
sensitization to be plausible based on the presence of a substituted
phenol group, with an EC3 of 1.7% (moderate sensitizer). Possible
mechanisms are: (i) Abstraction of the hydroxyl hydrogen atom to
generate a reactive phenolic radical species. Subsequent reaction with
skin proteins may occur directly or via hydrogen abstraction from a
protein thiol group. The resulting thiyl radical may then react with a
second phenolic radical species. (ii) Ring oxidation to yield the
corresponding catechol or hydroquinone. Further enzymatic or
auto-oxidation leads to the formation of electrophilic ortho- or
para-quinones capable of reacting directly with skin proteins via
A valid DPRA test was performed according to OECD 442C and GLP.
For the DPRA assayROC-118was dissolved in acetonitrile at 100 mM and
formed a clear solution by visual inspection.No co-elution of the test
item with synthetic peptides containing cysteine or lysine (SPCC or
SPCL) was observed, so that peptide depletion could be measured using
HPLC.In the cysteine reactivity assay the test item showed 39.6% SPCC
depletion, and in the lysine reactivity assay the test item showed 65.6%
SPCL depletion. However, since in both the SPCC and SPCL incubations
precipitation of the test item was observed after incubation, the
percentages of SPCC and SPCL depletion might be underestimated. The mean
of the SPCC and SPCL depletion was 52.6% and as a result ROC-118 was
classified in the “highreactivityclass” when using the Cysteine 1:10 /
Lysine 1:50 prediction model.
valid KeratinoSensTMassay was performed according to OECD
442D and GLP. For the KeratinoSensTMassayROC-118was
dissolved/suspended in dimethylsulfoxide to a final concentration of 200
100-fold dilution in DMEM (Dulbecco Modified Eagle Medium) of the200,
100 and 50 mMstocks
in dimethylsulfoxide formed a non-homogeneous solution and were
therefore not suitable to test. The
100-fold dilution of the 12.5 mM dimethylsulfoxide stock formed a
homogeneous solution (slight precipitation).The
dimethylsulfoxide stock and spike solutions were diluted 100-fold with
exposure medium resulting in test concentrations of0.060
– 125 µM.
The test item precipitated at the highest dose level tested.Two
independent experiments were performed.The
test item showed toxicity with an IC30of 40µMin
experiment 1 and no toxicity was observed in experiment 2.A
biologically relevant, dose-related induction of the luciferase activity
(EC1.5values of 0.90µMand
experiment 1 and 2, respectively) was measured in both experiments.The
maximum luciferase activity induction (Imax) was 68.36-fold
and 59.21-fold in experiment 1 and 2 respectively. In conclusion,ROC-118is
concluded as positive in the KeratinoSensTMassay, since
positive results (>1.5-fold induction) were observed at test
concentrations <1000 µM with a cell viability of >70% compared to the
Based on a positive DEREK NEXUS assessment, a positive DPRA assay
and a positive KeratinoSensTMassay, ROC-118 is concluded to
be a skin sensitizer. Performance of an additional in vitro assay
addressing the activation of dendritic cells would not yield additional
information if the test would give a negative or positive result. DEREK
NEXUS predicts a potency for skin sensitization for several groups
present in the main component and impurity 1; the EC3 for the phenyl
ester group and the alpha,beta-unsaturated ester group would lead to a
classification of 1B, while the predicted EC3 for a substituted phenol
in impurity 1 would lead to a classification of 1A. Based on these data
it cannot be excluded that ROC-118 will fall into category 1A. To
determine the correct classification further testing is needed. At the
moment only in vivo testing is available to determine potency,
and an LLNA study was therefore performed.
A Local Lymph Node Assay was performed, according to OECD guideline 429
and GLP principles, to asses the skin sensitising potential of ROC-118.
Three groups of five female mice were treated with test item
concentrations of 5, 10 or 25% w/w, these concentrations were selected
based on the results of a pre-screen test. Five vehicle control animals
were similarly treated with the vehicle (dimethylformamide) alone.
The test item was applied on three consecutive days, by open application
on the ears of the animals. Three days after the last exposure, all
animals were injected with 3H-methyl thymidine and after five hours the
draining (auricular) lymph nodes were excised and pooled for each
animal. After precipitating the DNa of the lymph node cells,
radioactivity measurements were performed. The activity was expressed as
the number of disintegrations per minute (DPM) and a stimulation index
(SI) was calculated for each concentration group. All auricular lymph
nodes of the animals of both the treatment groups and control groups
were considered normal in size. No macroscopic abnormalities of the
surrounding area were noted for any of the animals. The SI values
calculated for the test item concentrations 5, 10 and 25% were 0.9, 1.4
and 1.3, respectively. Since the test item did not induce a SI>3 when
tested up to 25%, it is not considered to be a skin sensitizer.
to the DEREK prediction, no metabolism is needed for the main component
(94%) to exert its sensitizing activity. Although the in chemico and in
vitro test predicted ROC-118 to be a skin sensitizer, the LLNA study
showed ROC-118 to be not sensitizing. The substance has a high molecular
weight (789 g/mol), a measured water solubility of <0.002 mg/L, log Pow
of 5.1 and is not expected to have surfactant-like properties. Based on
physchem parameters, the substance is not expected to penetrate the skin
significantly. The positive results from the in chemico/in vitro tests
may be due to the fact that penetration of the skin is not included in
these tests. As the LLNA is an in vivo test and there are no indications
that the substance does not fall within the applicability domain of the
test, the result from this test is considered to be conclusive: ROC-118
is not a skin sensitizer.
Based on the studies available for skin sensitization, ROC-118 is
considered to be no skin or respiratory sensitizer. ROC-118 does not
have to be classified according to Regulation 1272/2008 and amendments.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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