N,N-bis(2-hydroxyethyl)glycine

Skin sensitisation

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

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

Test material information

Constituent 1

Reference substance name:

N,N-bis(2-hydroxyethyl)glycine

EC Number:

205-755-1

EC Name:

N,N-bis(2-hydroxyethyl)glycine

Cas Number:

150-25-4

Molecular formula:

C6H13NO4

IUPAC Name:

N,N-bis(2-hydroxyethyl)glycine

impurity 1

Reference substance name:

N-(2-hydroxyethyl)ethylenediaminetriacetic acid

EC Number:

205-759-3

EC Name:

N-(2-hydroxyethyl)ethylenediaminetriacetic acid

Cas Number:

150-39-0

Molecular formula:

C10H18N2O7

IUPAC Name:

N-{2-[bis(carboxymethyl)amino]ethyl}-N-(2-hydroxyethyl)glycine

impurity 2

Reference substance name:

Not specified

IUPAC Name:

Not specified

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Test material form:

solid: crystalline

Details on test material:

Appearance: white crystalline powder
Storage conditions: at room temperature protected from light

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

In chemico test system

Details on the 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%

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: SPCC mean percentage

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Parameter:

other: SPCL mean percentage

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

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 C_water, 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.