[2-(2-methoxyethoxy)ethoxy]acetic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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)

Test material

Test material information

Constituent 1

Reference substance name:

[2-(2-methoxyethoxy)ethoxy]acetic acid

EC Number:

240-162-1

EC Name:

[2-(2-methoxyethoxy)ethoxy]acetic acid

Cas Number:

16024-58-1

Molecular formula:

C7H14O5

IUPAC Name:

2-[2-(2-methoxyethoxy)ethoxy]acetic acid

Test material form:

other: liquid: Clear colourless

In chemico test system

Details on the study design:

Instruments and Devices
Usual laboratory equipment and the following instruments were used in this study:
- Analytical and precison scales
- Volumetric measurement tools
- pH-meter
- Standard laboratory glassware
- Incubation chamber capable of holding 25 ± 2.5 °C

HPLC system:
Designation: HPLC_4
Components:
Degasser G1322A
Quaternary pump G1311A
Autosampler G1313A
Column compartment G1316A
UV/VIS-Detector DAD G1315A
Manufacturer: Agilent Technologies
Software: CHROMELEON 6.80 SR15b Build 4981
An ACE Excel SuperC18 150x3 mm column with 3 μm particles was used. This column was used instead the Agilent Zorbax SB-C18 column recommended in the OECD 442C guideline because it delivers substantially better peak shape for the peptides.

Peptides with ≥ 95 % purity, syntheseized by Genecust, Dudelange, Luxemburg, were used.
Sequence Cys-Peptide (Cysteine): Ac-RFAACAA-COOH (MW = 751.9 g/mol)
Sequence Lys-Peptide (Lysine): Ac-RFAAKAA-COOH (MW = 776.2 g/mol)

Chemicals
- Water for chromatography
- Demineralized water
- Acetonitrile for chromatography
- Trifluoroacetic acid
- Isopropanol
- Acetone
- Dimethylsulfoxide

Positive control
Positive controls are treated identically as the test item. The following positive controls were used:
- Cinnamaldehyde (CAS 104-55-2, food grade ≥95 %) used as 100 mM solution in acetonitrile for the cysteine peptide, Depletion range 60.8 – 100 %
- 2,3-Butanedione (CAS 431-03-8, >97 %) will be used as 100 mM solution in acetonitrile for the lysine peptide, Depletion range 10 – 45 %
As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments performed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione is used as positive control showing midrange depletion for the lysine peptide.

Solvent controls
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution are prepared in triplicate (Sets A, B1, B2 and C, total 12 samples per peptide). Set A is analysed together with the peptide calibration standards, sets B1, B2 and C are incubated with the samples. Sets B1 and B2 are analysed at the start and end of the analysis sequence and are used as stability control for the peptide over the total analysis time. Set C is analysed together with the samples and is used for calculation of the peptide depletion.

Acceptance criteria
- The standard calibration curve should have an r² > 0.99
- The measured values of solvent control samples of sets A and C should be 0.5 ± 0.05 mM
- The variation coefficient (relative standard deviation, RSD) of measured values of the nine samples from sets B1, B2 and C should be < 15 %
- The mean peptide depletion value for the positive control cinnamic aldehyde should be 60.8 % - 100 % for the Cys-Peptide and 40.2 % - 69.0 % for the lysine peptide with a maximum standard deviation (SD) of < 14.9 % for the Cys-Peptide and < 11.6 % for the Lys-Peptide. If one of the acceptance criteria is not fulfilled, the test is repeated. If the result is unambiguous even though one of the criteria is not met, the test may be considered valid by the study director, but justification must be given.
If the mean percent depletion falls in the range of 3% to 10% for the cysteine 1:10/lysine 1:50 prediction model or the cysteine percent depletion falls in the range of 9% to 17% for the cysteine 1:10 prediction model, a second run should be considered, as well as a third one in case of discordant results between the first two runs.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: mean of three runs

Parameter:

other: mean peptide depletion of both peptides (%)

Value:

1.88

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Run / experiment:

other: mean of three runs

Parameter:

other: Cys-Peptide depletion [%]

Value:

3.62

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Run / experiment:

other: mean of three runs

Parameter:

other: Lys-Ppetide depletion [%]

Value:

0.14

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Eight out of ten proficiency chemicals listed in the guideline were tested successfully. The results confirmed the classification reported in the OECD guideline (LAUS reference study 201704R875).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: yes
- Acceptance criteria met for positive control: yes

Applicant's summary and conclusion

Interpretation of results:

other: study cannot be used for classification alone

Conclusions:

The test item shows only minimal reactivity towards cysteine and lysine containing peptides and therefore no potential to cause skin sensitization.

Executive summary:

This study was performed in order to estimate the skin sensitisation potential of the test item using a peptide model. The direct peptide reactivity assay (DPRA) is an in chemico assay to quantify the reactivity of the test item towards cysteine and lysine containing peptides. This reactivity is related to the skin sensitisation potential. To quantify the sensitisation potential, the depletion of the cysteine and lysine containing peptides caused by known amounts of the test item is measured using HPLC. The assay is used for supporting the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 B is not possible. In this assay, the test item as well as the positive control (2,3-Butanedione) and the solvent control were tested in triplicate. 2,3-Butanedione shows a midrange depletion for the lysine peptide. As a result for the test item, the mean peptide depletion percentage was 3.62 for the Cys-Pepetide and 0.14 for the Lys-Peptide. The mean percentage of depletion of both peptides was 1.88, therefore predicting only minimal reactivity towards cysteine and lysine containing peptides and no potential to cause skin sensitization based on this reactivity.