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Administrative data

Description of key information

A DEREK, DPRA and keratinosens assay were performed.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
Dec 2016
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.
Reference:
Composition 0
Principles of method if other than guideline:
A DEREK assessment was performed.
GLP compliance:
no
Justification for non-LLNA method:
Since 11 oktober 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.
Test material information:
Composition 1
Specific details on test material used for the study:
Smiles: OCCN(CC(O)=O)CCO
Key result
Parameter:
other: prediction on skin sensitisation
Remarks on result:
no indication of skin sensitisation
Remarks:
DEREK predicted the substance to be not skin sensitising
Interpretation of results:
GHS criteria not met
Conclusions:
DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitisation for the test item. Bicin is predicted to be not sensitising to the skin.
Executive summary:

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitisation for the test item. Bicin is predicted to be not sensitising to the skin.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Dec 2016
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.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
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.
Test material information:
Composition 1
Specific details on test material used for the study:

Chemical name: 2-[bis(2-hydroxyethyl)amino]acetic acid
CAS number: 150-25-4
Molecular weight: 163.17 g/mol
Test item handling: use amber glassware or wrap container in aluminium-foil
Stability at higher temperatures: yes, max. temperature 40 °C, max. duration 2 days
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:

Test item preparation: No correction was made for the purity/composition 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 solvent was evaluated: acetonitrile (ACN) and water.
Test item stock solutions were prepared freshly for each reactivity assay.
For the cysteine reactivity assay 19.36 mg of the test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1186 μL water 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. For the lysine reactivity assay 24.33 mg of the test item was pre-weighed into a clean amber vial and dissolved, just before use, in 1491 μL water 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) and lysine (lys) reactivity assay.

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.
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
Positive control: cinnamic aldehyde
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.
Prior to HPLC-PDA analysis the samples were visually inspected. None of the samples showed precipitation.
Analysis: All samples were analyzed according to the HPLC-PDA method presented in Table 20 (APPENDIX 3) in the report. The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 21 (APPENDIX 4) in the report.
The time between the first injection (= RCcysB-1 or RClysB-1) and the last injection of the sequence did not exceed 30 hours.
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%
Key result
Parameter:
other: SPCC mean percentage
Value:
0
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Parameter:
other: SPCL mean percentage
Value:
0.4
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Interpretation of results:
GHS criteria not met
Conclusions:
It can be concluded that this DPRA test is valid, and that the test substance was negative in the DPRA and is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Executive summary:

In this study the reactivity of Bicin (CAS No. 150-25-4) 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 sensitisers and non-sensitisers.

Following 24 hours of incubation of Bicin 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 a prediction model.

Milli-Q water was found to be an appropriate solvent to dissolve Bicin, and was therefore used in this DPRA study.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and Cwater, 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 Bicin, were within the acceptability criteria for the DPRA assay.

No co-elution of the test item with SPPC or SPCL was observed.

In the cysteine reactivity assay Bicin showed 0.0% SPCC depletion, and in the lysine reactivity assay Bicin showed 0.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.2%. As a result Bicin was classified in the “no or minimal reactivityclass” when using the Cysteine 1:10/Lysine 1:50 prediction model. Therefore, Bicin was considered to be negative in the DPRA.

It can be concluded that this DPRA test is valid, and that bicin was negative in the DPRA and is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Jan - Feb 2017
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.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Test material information:
Composition 1
Specific details on test material used for the study:
Test item handling: Use amber glassware or wrap container in aluminum-foil
Stability at higher temperatures: Yes, maximum temperature: 40 °C, maximum duration: 2 days
Chemical name (IUPAC), synonym or trade name: 2-[bis(2-hydroxyethyl)amino]acetic acid
CAS Number: 150-25-4
Molecular weight: 163.17
pH 4-5 at concentration of 10%
Details on study design:
No correction was made for the composition/purity of the test item. The compound was protected from light (amber coloured glassware was used).
The test item was dissolved in DMSO to a final concentration of 200 mM. The compound formed a translucent suspension at 200 mM. From the stock 11 spike solutions in DMSO were prepared (2-fold dilution series): 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, 0.391, 0.195 and 0.0977 mM. The test item formed a suspension at 100 mM and dissolved 50 mM and lower.
The stock and spike solution were diluted 25-fold with exposure (final concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95 and 0.977 µM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. Two independent experiments were performed. All formulations formed a clear solution.
Positive control: ethylene dimethacrylate glycol (7.81 to 250 uM)

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±1.0oC in the presence of 5% CO2. In total 2 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 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:
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 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 7.81 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 2.69 and the EC1.5 23.3 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.62 and the EC1.5 26.7 µM.
Key result
Run / experiment:
experiment 1
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: Imax 0.84-fold
Key result
Run / experiment:
experiment 2
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: Imax 1.39-fold
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 (23.3 µM and 26.7 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.69-fold and 3.62-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% (9.5% and 7.2% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Bicin showed no toxicity (no IC30 and IC50 value) and 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 0.84-fold and 1.39-fold in experiment 1 and 2 respectively. Bicin is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations of ≥1000 µM with a cell viability of >70% compared to the vehicle control.
Interpretation of results:
GHS criteria not met
Conclusions:
The KeratinoSensTM assay is valid and the test substance is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Executive summary:

A KeratinoSensTM assay was performed with bicin according to OECD 442D and GLP. Bicin was dissolved in dimethylsulfoxide 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 of0.98 – 2000 µM (2-fold dilution series). Two independent experiments were performed. No precipitation was observed at any of the test concentrations. The test conditions were adequate and that the test system functioned properly.

Bicin showed no toxicity (no IC30and IC50value) and no biologically relevant induction of the luciferase activity (no EC1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 0.84-fold and 1.39-fold in experiment 1 and 2 respectively. Bicin is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations of ≥1000 µM with a cell viability of >70% compared to the vehicle control.

Finally, it is concluded that the KeratinoSensTM assay is valid and that Bicin is classified as negative(noactivation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

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

DPRA:

In this study the reactivity of Bicin (CAS No. 150-25-4) towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined toassign the test chemical to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Following 24 hours of incubation of Bicin 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 a prediction model.

Milli-Q water was found to be an appropriate solvent to dissolve Bicin, and was therefore used in this DPRA study.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and Cwater, 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 Bicin, were within the acceptability criteria for the DPRA assay.

No co-elution of the test item with SPPC or SPCL was observed.

In the cysteine reactivity assay Bicin showed 0.0% SPCC depletion, and in the lysine reactivity assay Bicin showed 0.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.2%. As a result Bicin was classified in the “no or minimal reactivityclass” when using the Cysteine 1:10/Lysine 1:50 prediction model. Therefore, Bicin was considered to be negative in the DPRA.

It can be concluded that this DPRA test is valid, and that bicin was negative in the DPRA and is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

A KeratinoSensTM assay was performed with bicin according to OECD 442D and GLP.Bicin was dissolved in dimethylsulfoxide 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 of0.98 – 2000 µM (2-fold dilution series). Two independent experiments were performed. No precipitation was observed at any of the test concentrations. The test conditions were adequate and that the test system functioned properly.

Bicin showed no toxicity (no IC30and IC50value) and no biologically relevant induction of the luciferase activity (no EC1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 0.84-fold and 1.39-fold in experiment 1 and 2 respectively. Bicin is classified as negative in the KeratinoSensTMassay since negative results (<1.5-fold induction) were observed at test concentrations of ≥1000 µM with a cell viability of >70% compared to the vehicle control.

Finally, it is concluded that the KeratinoSensTMassay is valid and that Bicin is classified as negative(noactivation of theantioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes)under the experimental conditions described in this report.

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitisation for the test item. Bicin is predicted to be not sensitising to the skin.

In conclusion, based on the results from the DEREK assessment, the DPRA assay and the KeratinoSensTMassay, bicin is not a skin sensitizer. (see attached report in Ch13).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

As the substance is no skin sensitizer, it is likely that the substance is not a respiratory sensitiser according to Guidance on information requirements R7a.

Justification for classification or non-classification

Based on the data available bicin does not have to be classified for skin or respiratory sensitization according to Regulation 1272/2008 and amendments.