Registration Dossier

Administrative data

Description of key information

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.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
QSAR
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Principles of method if other than guideline:
A DEREK assessment was performed. In addition to the main constituent of the substance, two impurities were evaluated.
GLP compliance:
no
Justification for non-LLNA method:
Since 11 October 2016 it is legally required to consider all available information for the endpoint skin sensitisation and to use a non in vivo test strategy based on in chemico, in silico and in vitro skin tests combined with a WoE. An in vivo test (LLNA) is only allowed as last resort. DEREK results are adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitisation.
Key result
Parameter:
other: prediction on skin sensitisation
Run / experiment:
Main component
Remarks on result:
positive indication of skin sensitisation
Remarks:
DEREK predicted the main component to be sensitizing (plausible)
Key result
Parameter:
other: prediction on skin sensitisation
Run / experiment:
Impurity 1
Remarks on result:
positive indication of skin sensitisation
Remarks:
DEREK predicted impurity 1 to be sensitizing to the skin (plausible)
Key result
Parameter:
other: prediction on skin sensitisation
Run / experiment:
Impurity 2 (same as main component)
Remarks on result:
positive indication of skin sensitisation
Remarks:
DEREK predicted impurity 2 to be sensitizing.
Other effects / acceptance of results:
-Prediction for main constituent: 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 the Michael addition applies.

- Prediction for impurity 1: DEREK NEXUS predicts an 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 Michael addition.

- Prediction for impurity 2: positive, based on the presence of a phenyl ester group (see main constituent).
Interpretation of results:
study cannot be used for classification
Remarks:
(study is part of a weight of evidence approach and is not used for classification on its own)
Conclusions:
DEREK NEXUS version 5.0.2 predicted the main constituent and both impurities of ROC-118 to be sensitising to the skin (plausible).
Executive summary:

DEREK NEXUS version 5.0.2 predicted the main constituent and both impurities of ROC-118 to be sensitising to the skin (plausible).

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22 June 2017 - 04 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd 3 November 2015
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
A validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as one of the non-animal tests to be performed as part of weight of evidence.
Details on study design:
TEST ITEM PREPERATION
A correction factor of 1.064 was applied for the purity of the test item. Solubility of the test item was assessed before performing the DPRA assay. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), MQ/ACN (1:1, v/v), isopropanol, acetone, acetone/ACN (1:1, v/v) and dimethylsulfoxide (DMSO)/ACN (1:9, v/v).
Test item stock solutions were prepared freshly for each reactivity assay.
For the cysteine and lysine reactivity assay, respectively, 127.38 mg and 88.45 mg of the test item were pre-weighed into a clean amber glass vial and dissolved, just before use, in 1518 μL and 1054 μL ACN, respectively, to obtain 100 mM solutions. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assays.

TEST SYSTEM
Synthetic peptides containing cysteine (SPCC) (Ac- RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight of SPCC is 750.9 g/mol, and 775.9 g/mol for SPCL. The peptides were stored in the freezer (<-15°C) for a maximum of 6 months.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Calibration curve SPCC and SPCL: according to guideline
- Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler, in the dark, and incubated at 25 ± 2.5°C for 24 ± 2 hours. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.5 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.
- Analysis: All samples were analyzed according to the HPLC-PDA method presented in Table 1 ('Other information on methods and materials'). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 ('Other information on materials and methods').

POSITIVE CONTROL: Cinnamic aldehyde, kosher
- Purity: 98.4%
- Batch: MKBP1014V
- Expiry of batch: 31 May 2018

DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration, and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion = [1-(Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))]*100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample a ratio in the range of 90%< mean area ratio of control samples <110% gives a good indication that co-elution has not occurred.

DATA INTERPRETATION (see also 'Other information on materials and method')
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.
Key result
Parameter:
other: SPCC mean percentage
Run / experiment:
Cysteine Reactivity Assay
Value:
39.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
CV between reference controls: 2.3%
Positive controls validity:
valid
Remarks:
Mean percentage SPCC: 75.9% ±1.3%
Remarks on result:
other: SD: 1.2%
Key result
Parameter:
other: SPCL mean percentage
Run / experiment:
Lysine Reactivity Assay
Value:
65.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
CV between reference controls:2.6%
Positive controls validity:
valid
Remarks:
Mean percentage SPCL: 57 ± 3.1%
Remarks on result:
other: SD: 4.4%

Table 4 Acceptability of the DPRA assay

 

Cysteine reactivity assay

Lysine reactivity assay

Acceptability criteria

Results for SPCC

Acceptability criteria

Results for SPCL

Correlation coefficient (r2) standard calibration curve

>0.99

0.994

>0.99

0.996

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05

0.485 ± 0.027

0.50 ± 0.05

0.516 ± 0.020

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

0.488*

0.50 ± 0.05

0.513 ± 0.007

CV (%) for RC samples

B and C

<15.0

2.3*

<15.0

2.6

Mean peptide depletion cinnamic aldehyde (%)

60.8-100

75.9

40.2-69.0

57.0

SD of peptide depletion cinnamic aldehyde (%)

<14.9

1.3

<11.6

3.1

SD of peptide depletion for Perfluoro methoxy dioxole (%)

<14.9

1.2

<11.6

4.4

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.

Table 5 SPCC and SPCL depletion and reactivity classification forPerfluoro methoxy dioxole

Test item

SPCC depletion

SPCL depletion

Mean of SPCC and SPCL depletion

Reactivity class

Mean

± SD

Mean

± SD

Cysteine 1:10 / Lysine 1:50 prediction model

ROC-118

39.6%

±1.2%

65.6%

±4.4%

52.6%

High reactivity

Interpretation of results:
study cannot be used for classification
Remarks:
Study is part of a weight of evidence approach and is not used for classification on its own.
Conclusions:
ROC-118 was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Executive summary:

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 sensitisers.

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 model.

 

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.

 

In 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 the DPRA.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
21 July 2017 - 11 August 2017
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)
Version / remarks:
February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The KeratinoSensTM assay is recommended in international guidelines (e.g. OECD) for substitution of animal testing and mentioned in the ECHA guidance as the in vitro test to be performed as part of weight of evidence.
Specific details on test material used for the study:
- pH (1% in water, indicative range): 5.12-4.78
- Solubility in vehicle (DMSO): 1.5 g/mL
Details on study design:
TEST ITEM PREPARATION
A correction factor of 1.064 was used to correct for the composition/purity of the test item. The test item was dissolved in DMSO to a final concentration of 200 mM. The 100-fold dilution in DMEM of 200, 100, 50 and 25 mM formed a non-homogeneous solution and was therefore not suitable to test. The 100-fold dilution of the 12.5 mM DMSO stock formed a homogeneous solution (slight precipitation). This concentration was selected as highest concentration for the main assay.

The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49, 0.24, 0.12 and 0.060 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. Precipitation was observed at the start and end of the incubation period in the 96- well plates at the highest concentration.

Test item concentrations were used within 3 hours after preparation.

CONTROL ITEMS
- Positive control: ethylene dimethacrylate glycol (tested in triplicate)
*Purity: 98%
*Batch: SHBF2397V
*Amount used: 0.78 to 25 mM in DMSO, diluted so that the final concentration ranged from 7.8 to 250 μM (final concentration DMSO of 1%)
- Negative control: eighteen wells per plate of a solvent control of 1% DSMO were tested
- Blank control: on each plate three bank wells were tested (no cells and no treatment)

TEST DESIGN
- Test system: A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

- Plating of cells: For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator.

- Treatment of cells: The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1°C in the presence of 5% CO2. Initially experiment 1 was rejected due to technical reasons, so the experiment was repeated.

- Luciferase activity measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time one second).

- Cytotoxicity assessment: For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured with the TECAN Infinite® M200 Pro Plate Reader.

DATA ANALYSIS: according to guideline

Interpretation of results as positive:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations <1000 µM or 200 μg/mL should be considered as inconclusive.

Acceptance criteria:
•The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
•The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
•Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
Positive control results:
Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.31 and the EC1.5 51 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.35 and the EC1.5 46 µM.
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
68.36
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
Imax: 2.31; EC1.5: 51 μM
Remarks on result:
other: EC1.5: 0.90 μM
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
59.21
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
Imax: 2.35; EC1.5: 46 μM
Remarks on result:
other: EC1.5: 0.70 μM
Other effects / acceptance of results:
Both experiments passed the acceptance criteria:
•The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
•The EC1.5 of the positive control was between 5 and 125 μM in experiment 2 and 3 (51 and 45.6 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.30-fold and 2.35-fold in experiment 1 and 2, respectively).
•Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (7.1%, and 5.5% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test item showed toxicity in the first experiment (IC30 value of 40 μM).
Interpretation of results:
study cannot be used for classification
Remarks:
Study is part of a weight of evidence approach and is not used for classification on its own.
Conclusions:
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 KeratinoSensTM 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.
Executive summary:

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 criteria.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

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 Michael addition.

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.

A valid KeratinoSensTMassay was performed according to OECD 442D and GLP. For the KeratinoSensTMassayROC-118was dissolved/suspended in dimethylsulfoxide to a final concentration of 200 mM. The 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 0.70µMin 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 vehicle control.

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.

According 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.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

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.