Registration Dossier

Administrative data

Description of key information

A DEREK assessment, DPRA assay and KeratinoSensTM assay were performed. Based on these data, it is concluded that HYDROPIP-NR is not a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
In silico assessment
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
2018
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.
Principles of method if other than guideline:
An in silico assessment was performed with DEREK NEXUS version 6.0.0
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 sensitzation
Run / experiment:
HYDROPIP-NR
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
DEREK NEXUS version 6.0.0 did not yield any alerts for skin sensitization for the test item. However, the query structure contains features that have been observed in sensitizers that do not match skin sensitization structural alerts in Derek (misclassified). The relationship between this feature and skin sensitisation may be coincidental or contributory. As the query, structure does not match any structural alerts or examples for skin sensitization in DEREK. HYDROPIP-NR is predicted to be not sensitizing to the skin.
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:
HYDROPIP-NR is predicted to be not sensitizing to the skin.
Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 January 2018 - 30 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 PREPERATION
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. 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 solvent was evaluated: acetonitrile (ACN). Test item stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay 48.59 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1715 μL ACN to obtain a 100 mM solution. 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 assay, respectively.

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, 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. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 25 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
- 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 Table 3 '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:
1.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
CV between reference controls: 5.0%
Positive controls validity:
valid
Remarks:
Mean percentage SPCC: 79.3% ± 5.3%.
Remarks on result:
other: SD: 1.5%
Parameter:
other: SPCL mean percentage
Run / experiment:
Lysine Reactivity Assay
Vehicle controls validity:
not applicable
Negative controls validity:
not valid
Remarks:
CV between reference controls: 38.5%
Positive controls validity:
valid
Remarks:
mean percentage SPCL: 68.1% ± 3.5%
Remarks on result:
other: Co-elution of the test item with SPCL, Therefore the depletion could not be determined
Other effects / acceptance of results:
Co-elution occured between the test item and SPCL.
See table 4 and 5 in " any other information on results incl. table" for results and acceptability of DPRA

Table 4: 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

0.997

>0.99

0.998

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05 

0.507 ± 0.007

0.50 ± 0.05

0.496 ± 0.001

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

0.505 ± 0.008

0.50 ± 0.05

0.742 ± 0.293

CV (%) for RC samples B and C

<15.0

5.0

<15.0

38.5

Mean peptide depletion cinnamic aldehyde (%)

60.8-100

79.3

40.2-69.0

68.1

SD of peptide depletion cinnamic aldehyde (%)

<14.9

5.3

<11.6

3.5

SD of peptide depletion for the test item (%)

<14.9

1.5

<11.6

NA

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation; NA = Not Applicable

Table 5: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item 

SPCC depletion 

SPCL depletion

DPRA prediction and reactivity classification

Mean

± SD

Cysteine 1:10 prediction model

HYDROPIP-NR

1.2%

±1.5%

Co-elution

Negative: No or minimal reactivity

SD = Standard Deviation.

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:
HYDROPIP-NR was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 prediction model.
Executive summary:

A Direct Peptide Reactivity Assay (DPRA) was performed according to OECD TG 422C and in compliance with GLP. HYDROPIP-NR was dissolved in acetonitrile at 100 mM. Upon preparation as well as after incubation of the test item with synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) no precipitate was observed. In the cysteine reactivity assay the test item showed 1.2% SPCC depletion. Co-elution was observed in the lysine reactivity assay and therefore SPCL depletion could not be calculated. Based on the results, HYDROPIP-NR was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 prediction model and was considered to be negative in the DPRA.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12 December 2017 - 26 January 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 2000 μM was selected as highest concentration for the main assay. This dose was the highest dose required in the current guideline.

In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM (clear colorless solutions). The stock was sonicated in the first and third experiment (Time: 7 and 9 min; Temp.: 22.0 – 28.0 °C and 18.5 – 20.5 °C; in experiment 1 and 3, respectively). 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 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate.

Test item concentrations were used within 1.5 hour 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. 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. The passage number used was P+12 in experiment 1, P+7 in experiment 2 and P+9 in experiment 3.

- 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. 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±1.0°C in the presence of 5% CO2. In total 3 valid experiments were performed.

- 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 a microplate reader to assess the quantity of luciferase (integration time two seconds).

- 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 and subsequently 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 at 570 nm with a microplate reader.

The KeratinoSensTM test is considered acceptable if it meets the following 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 negative (solvent) control DMSO should be below 20% in each repetition whichconsists of 18 wells tested. If the variability is higher, results should be discarded.

A KeratinoSensTM 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 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.

Positive control results:
Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.05 and the EC1.5 72 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 4.27 and the EC1.5 60 µM.
Experiment 3: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 5.76 and the EC1.5 10 µM.
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
2.13
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: EC1.5: 1370 μM
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
7.32
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: EC1.5: 782 μM
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 3
Value:
2.19
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: EC1.5: 1337 μM
Other effects / acceptance of results:
All 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 (72 μM, 60 μM and 10 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.05-fold, 4.27-fold and 5.76-fold in experiment 1, 2 and 3, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (8.9%, 9.0% and 9.1% in experiment 1, 2 and 3,
respectively).

CYTOTOXICITY:
The test item showed toxicity.

Experiment 1:
The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.

Experiment 2:
IC30: 1304 µM
IC50: 1970 µM
* At 500 μM a slight decrease in viability was observed (viability 63.7%), but since at 1000 μM there was no decrease in viability this decrease was considered to be caused by variation in the data and not relevant for the toxicity endpoint.

Experiment 3
IC30: 1710 µM
IC50: Not reached

PRECIPITATION:
No precipitation was observed at the start and end of the incubation period in the 96-well plates in all experiments.

Three independent experiments were performed. A third experiment was performed since the first 2 experiments were not concordant.

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:
HYDROPIP-NR is classified as negative in the KeratinoSensTM assay since negative results (no relevant induction) were observed in 2 out of 3 experiments.
Executive summary:

A valid KeratinoSensTM assay was performed according to OECD TG 442D and in accordance with GLP principles. HYDROPIP-NR was dissolved in DMSO to a final concentration of 200 mM. The cells were incubated with HYDROPIP-NR in a 2-fold dilution series (0.98μM – 2000μM) for 48 hours in the experiments. No precipitation was observed at the start and end of the experiments. In the first experiment no cytotoxicity was observed. An induction of the luciferase activity (EC1.5 value of 1370μM) was measured and the maximum luciferase activity induction (Imax) was 2.13-fold. Since the EC1.5 is 1000μM the outcome of the first experiment is negative. In the second experiment, the test item showed toxicity (IC30 and IC50 of 1304μM and 1970μM, respectively). An induction of the luciferase activity (EC1.5 value of 782μM) was measured and the Imax was 7.32-fold. Since the EC1.5 is <1000μM the outcome of the second experiment was positive. Since these outcomes were contradictory, a third experiment was performed. In the third experiment cytotoxicity was observed (IC30 value of 1710μM). An induction of the luciferase activity (EC1.5 value of 1337μM) was measured and the Imax was 2.19-fold. Since the EC1.5 is 1000μM the outcome of this run was negative. Based on the results HYDROPIP-NR is classified as negative in the KeratinoSensTM assay since negative results were observed in 2 out of 3 experiments.

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

The DEREK assessment predicted HYDROPIP-NR to be not sensitizing to the skin. HYDROPIP-NR did not significantly deplete SPCC in the DPRA and therefore the DPRA outcome was negative. Depletion of SPCL could not be measured due to co-elution with the test item. In the KeratinoSensTM assay activation of keratinocytes was observed in all three experiments. In 2 out of 3 runs significant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway was observed at concentrations higher than 1000μM. Induction at these dose levels are not considered relevant for the skin sensitization endpoint and therefore the overall outcome of the KeratinoSensTM assay was negative. Based on the results, it was concluded that HYDROPIP-NR is not a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

HYDROPIP-NR does not need to be classified for skin sensitization according to Regulation (EC) No 1272/2008 and related amendments.