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Description of key information

A positive DEREK, a positive DPRA and an inconclusive Keratinosens assay were obtained. Based on a weight of evidence approach, the substance was considered to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
In Silico
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
Justification for type of information:
An in silico assesment is mentioned in the ECHA guidance as one of the non-animal data that may be provided as weight of evidence.
Qualifier:
no guideline followed
Principles of method if other than guideline:
An in silico assessment was performed with DEREK NEXUS version 6.0.1.
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:
Run / experiment:
Test substance
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
DEREK NEXUS version 6.0.1 yielded an alert (equivocal) for the subtance for skin sensitization based on the presence of the substituted phenol group and predicted a LLNA EC3 of 0.18% (strong sensitizer) based on structurally similar compounds. As the analogues used for the calculation of EC3 had a similarity of 26-39%, mostly other groups than alkyl groups on the phenol ring, and skin sensitizing properties varied from non-sensitizing to extreme sensitizer, the predicted EC3 should be considered with caution, and is a worst case.
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 6.0.1 yielded an alert for the substance for skin sensitization based on the presence of the substituted phenol group and predicted a LLNA EC3 of 0.18% (strong sensitizer).
Executive summary:

DEREK NEXUS version 6.0.1 yielded an alert (equivocal) for the substance for skin sensitization based on the presence of a substituted phenol group.

Based on data on closest structurally-related substances, an EC3 for the substance of 0.18% (strong sensitizer) was predicted. As the analogues used for the calculation of EC3 had a similarity of 26-39%, the predicted EC3 should be considered with caution, and is a worst case.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 January 2018 - 18 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The 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 the in chemico test to be performed as part of weight of evidence.
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
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 PREPARATION
Solubility of the test substance in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test substance 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), ACN/MQ (1:1, v/v), isopropanol, acetone/ACN (1:1, v/v), dimethylsulfoxide (DMSO)/ACN (1:9, v/v) and methanol.
Test substance stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 56.95 mg of test substance was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1622 µL ACN/MQ (1:1, v/v) to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test substance was dissolved. The test substance, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

TEST SYSTEM
Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL. Rationale: Recommended test system in the international OECD guideline for DPRA studies.
Source: JPT Peptide Technologies GmbH, Berlin, Germany.
Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test substance samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 26 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.

POSITIVE CONTROL
Cinnamic aldehyde
- 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))]x100

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%
DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test substance. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see Table 1), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.
Parameter:
other: Mean SPCC depletion(%)
Run / experiment:
Cysteine Reactivity Assay
Value:
1.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
CV between reference controls: 1.6%
Positive controls validity:
valid
Remarks:
Mean percentage SPCC: 78.7% ±1.1%
Remarks on result:
other: SD: 1.0%
Parameter:
other: Mean SPCL depletion (%)
Run / experiment:
Lysine Reactivity Assay
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
CV between reference controls: 3.7%
Positive controls validity:
valid
Remarks:
Mean percentage SPCL: 59 ± 1.3%
Remarks on result:
other: SD: 0.0%
Other effects / acceptance of results:
Upon preparation of the SPCC test item samples, a precipitate was observed while no precipitate was observed in the SPCC test item samples after incubation.
Upon preparation as well as after incubation of the SPCL test item samples, a precipitate was observed.

See Table 2 & 3 "any other information on results incl. tables" for acceptibility criteria.

Table 2: Acceptability of the Direct Peptide Reactivity Assay (DPRA)

 

Cysteine reactivity assay

Lysine reactivity assay

Acceptability criteria

Results for SPCC

Acceptability criteria

Results for SPCL

Correlation coefficient (r2) standard calibration curve

>0.99

0.998

>0.99

0.997

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05

0.501 ± 0.003

0.50 ± 0.05

0.509 ± 0.010

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

0.496 ± 0.006

0.50 ± 0.05

0.518 ± 0.004

Mean peptide concentration RC-CACN/MQsamples (mM)

0.50 ± 0.05

0.495 ± 0.001

0.50 ± 0.05

0.497 ± 0.002

CV (%) for RC samples

B and C

<15.0

1.6

<15.0

3.7

Mean peptide depletion cinnamic aldehyde (%)

60.8-100

78.7

40.2-69.0

59.1

SD of peptide depletion cinnamic aldehyde (%)

<14.9

1.1

<11.6

1.3

SD of peptide depletion for the test substance (%)

<14.9

1.0

<11.6

0.0

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation

table 3: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification forthe Test Item

SPCC depletion

SPCL depletion

Mean of SPCC and SPCL depletion

DPRA prediction and  reactivity classification

Mean

± SD

Mean

± SD

Cysteine 1:10 / Lysine 1:50 prediction model

 

Test substance

1.8%

±1.0%

100.0%

±0.0%

50.9%

Positive: High reactivity

SD = Standard Deviation.

Interpretation of results:
other: Study cannot be used for classification independetly, but in a WoE for the end point Skin Sensitisation
Conclusions:
The test substance 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 the test substance 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.

 ACN/MQ (1:1, v/v) 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. 

Upon preparation of the SPCC test item samples, a precipitate was observed while no precipitate was observed in the SPCC test item samples after incubation. Upon preparation as well as after incubation of the SPCL test item samples, a precipitate was observed.

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 substance showed 1.8% SPCC depletion while in the lysine reactivity assay the test substance showed 100.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 50.9% and as a result the test substance was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Therefore, the test substance 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:
19 January 2018 - 09 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in vitro test to be performed as part of weight of evidence.
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
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.
Details on study design:
TEST ITEM PREPARATION
Based on a solubility test, a concentration of 250 μM in DMSO was selected as highest concentration for the main assay (limit of solubility).

In the main experiments the test item was dissolved in DMSO at 25 mM (colourless). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). 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 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49, 0.24 and 0.12 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution.

Test item concentrations were used within 3 hours after preparation.

CONTROL ITEMS
- Positive control: ethylene dimethacrylate glycol (tested in triplicate)
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: The KeratinoSens™ cell line, having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used. Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock.
- Plating of cells: For testing, cells were 80-90% confluent. The passage number used was P+10 in experiment 1 and P+12 in experiment 2.
- 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 items were added.The treated plates were covered with foil and then incubated for about 48 hours at 37±1.0°C in the presence of 5% CO2. In total 2 experiments were performed.
- Luciferase activity measurement: Plates withcell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).
-Cytotoxicity assessment: For the KeratinoSens 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. Cells were lysed and subsequently the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

DATA ANALYSIS: according to guideline

Interpretation:
A KeratinoSens prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the vehicle (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:
a) 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).
b) 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.
c) The average coefficient of variation of the luminescence reading for the vehicle (negative) 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 2.57 and the EC1.5 74 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.98 and the EC1.5 40 µM.
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
1.21
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: EC1.5: could not be calculated
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
1.45
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: EC1.5: could not be calculated
Other effects / acceptance of results:
Both tests 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 (74 μM and 40 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.57-fold and 2.98-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.4% and 7.4% in experiment 1 and 2, respectively).

CYTOTOXICITY:
The test item showed toxicity in both experiments

Experiment 1
IC30: 47 µM
IC50: 51 µM

Experiment 2
IC30: 56 µM
IC50: 67 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:
The test substance is classified in the Keratinosens assay as inconclusive since negative results were observed at test concentrations < 1000 μM (up to 250 μM) due to a limit in solubility.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

DEREK:

DEREK NEXUS version 6.0.1 yielded an alert for the test substance for skin sensitization based on the presence of the substituted phenol group. The test substance is predicted to be sensitizing to the skin (equivocal). DEREK NEXUS predicted a LLNA EC3 of 0.18% (strong sensitizer) based on results on most structurally similar compounds. As the analogues used for the calculation of EC3 had a similarity of 26- 39 %, mostly other groups than alkyl groups on the phenol ring, and skin sensitizing properties varied from non-sensitizing to extreme sensitizer, the predicted EC3 should be considered with caution, and is a worst case.

DPRA:

A valid DPRA test was performed according to OECD 442C and GLP. For the DPRA assay, the test substance was dissolved in acetonitrile/Milli-Q water (1:1, v/v) at 100 mM and formed a clear solution by visual inspection.No co-elution of the test item with SPCC or SPCL was observed.In the cysteine reactivity assay the test item showed 1.8% SPCC depletion, and in the lysine reactivity assay the test item showed 100% SPCL depletion. The mean of the SPCC and SPCL depletion was 50.9%. As a result, the test item was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model and was considered to be positive in the DPRA.

KERATINOSENS

A valid Keratinosens assay was performed according to OECD 442D and GLP. For the Keratinosens assay, the test item was dissolved in DMSO to a final concentration of 25 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 0.12 – 250 µM (2-fold dilution series). The highest test concentration was considered to be the limit of solubility. No precipitate was observed at any dose level tested. Two independent experiments were performed. The test item showed toxicity with an IC30 of 47 µM and an IC50 of 51 µM in experiment 1, and an IC30 of 56 µM and IC50 value of 67 µM in experiment 2. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.21-fold and 1.45-fold in experiment 1 and 2 respectively. In conclusion, the test substance classified as inconclusive in the Keratinosens assay, since negative results (<1.5-fold induction; noactivation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) were observed at test concentrations of <1000 µM in both experiments.

Based on a positive DEREK NEXUS assessment, a positive DPRA assay and an inconclusive Keratinosens assay, it can be concluded that the test substance has skin sensitizing properties, and is predicted to be a strong sensitizer based on DEREK EC3 prediction of 0.18%. Performance of an additional in vitro assay, U-Sens assay, addressing the activation of dendritic cells would not yield additional information if it was negative or positive. Therefore, further in vitro testing is considered not scientifically required. Based on the EC3 prediction related to 10 structurally similar compounds, classification of the test substance is possible, and no further in vivo testing is needed. As the analogues used for the calculation of EC3 had a similarity of 26 - 39%, mostly other groups than alkyl groups on the phenol ring, and skin sensitizing properties varied from non-sensitizing to extreme sensitizer, the predicted EC3 should be considered with caution, and therefore represents a worst case.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, the test substance is classified as a category 1 skin sensitizer and is required to be labelled with H317 (May cause an allergic skin reaction) according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).