Bis(2-ethylhexyl) malate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Jan 2018 to 11 Jan 2018

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:

05 Feb 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Pepditde reactivity Assay - DPRA

Justification for non-LLNA method:

This is a Non in vivo test and the test material is used in cosmetic ingredients. Regulation 1223/2009 Article 18 restricts the use of in vivo studies on these types of raw materials.

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) malate

EC Number:

260-070-5

EC Name:

Bis(2-ethylhexyl) malate

Cas Number:

56235-92-8

Molecular formula:

C20H38O5

IUPAC Name:

1,4-bis(2-ethylhexyl) 2-hydroxybutanedioate

Test material form:

liquid

Details on test material:

Identification: Dermol DOM
Appearance: Clear colourless to pale yellow liquid
Batch: P7677
Purity/Composition: >95%
Test item storage: At room temperature
Stable under storage conditions until: 31 December 2018 (retest date)
Additional information
Test Facility test item number: 209081/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Molecular structure: Not indicated
Molecular formula: C20H38O5
Molecular weight: 358.5

Specific details on test material used for the study:

Test Article Dermol DOM
CAS Number 56235-92-8
Storage 15 to 25°C, protected from light
Batch Number P7677
Expiration Date 26 August 2018
Purity >95%

In chemico test system

Details on the study design:

Objectives
The study was conducted to quantify the reactivity of the test materail towards model synthetic peptides containing either lysine or cysteine. The data is used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptides following incubation with the test article. Relative peptide concentration was measured by high performance liquid chromatography (HPLC) with UV detection. Cysteine and lysine peptide percent depletion (PPD) values were then calculated and used in a prediction model which allows assigning the test article to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Test Article Incubation
Each test solution was prepared at ratios of 1:10 and 1:50 with the cysteine and lysine stock solutions, respectively. The preparations were placed in an incubator set at 25°C or 24±2 hours. At the end of the incubation period all test article and co-elution
samples contained small particles, therefore all samples were centrifuged at 400 g for 5 minutes.

Analytical Method
The following HPLC conditions were applied:
Column: Agilent Zorbax SB-C18 2.1 mm x 100 mm, 3.5 µm or equivalent
Wavelength: 220 nm
Guard column: Phenomenex Security Guard c18 4 mm x 2 mm
Flow rate: 0.35 mL/min
Oven temperature: 30°C
Sample temperature: 25°C
Injection volume: 7 µL

Mobile Phase:
Phase A: 0.1% (v/v) of trifluoroacetic acid in MilliQ water
Phase B: 0.085% (v/v) of trifluoroacetic acid in acetonitrile

Gradient: Time (min) Phase A Phase B
0 90 10
10 75 25
11 10 90
13 10 90
13.5 90 10
20 90 10



Reference and Co-elution Controls
Reference controls were prepared for each peptide.
Reference Control A and B for each peptide were prepared by adding 750 µL of peptide stock solution to 250 µL of acetonitrile.
Reference Control C for cysteine was prepared by adding 750 µL of peptide stock solution to 200 µL of acetonitrile and 50 µL vehicle.
Reference Control C for lysine was prepared by adding 750 µL of peptide stock solution to 250 µL vehicle.
Reference Control A (in triplicate) was used to verify the HPLC system suitability prior to the analysis. Reference Control B (six replicates) was used to verify the stability of the reference controls over time and Reference Control C (in triplicate)
was used to verify that acetonitrile did not impact the percent peptide depletion.

Co-elution controls were prepared to detect possible co-elution of the test article with the peptides. A mixture of 750 µL of 100 mM Phosphate Buffer pH 7.5, 200 µL of acetonitrile and 50 µL of test article solution was used to detect possible co-elution of
the test article with cysteine. A mixture of 750 µL of 100 mM ammonium acetate buffer pH 10.2 and 250 µL of test article solution was used to detect possible co-elution of the test article with lysine.
Calibration Curves for Peptides
Calibration curves were prepared for each peptide using a range of concentrations from approximately 0.534 mM to 0.0167 mM (Standards 1 to 6).
Standard 1 for cysteine was prepared at approximatively 0.534 mM by dilution of 1600 µL of the peptide stock solution (0.667 mM) with 400 µL of acetonitrile.
Standards 2 to 6 for cysteine were prepared by serial dilution using dilution buffer
(20% acetonitrile in 100 mM Phosphate Buffer pH 7.5).
Standard 1 for lysine was prepared at approximatively 0.534 mM by dilution of 800 µL of the peptide stock solution (0.667 mM) with 200 µL of acetonitrile.
Standards 2 to 6 for lysine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM ammonium acetate buffer pH 10.2).
Samples of dilution buffer alone were also prepared.
Sample Analysis Sequence
The analysis sequence for each peptide was as follows:
System suitability Standard 1 Dilution buffer
Calibration standards and reference controls Standard 1
Standard 2
Standard 3
Standard 4
Standard 5
Standard 6
Dilution Buffer
Reference Control A, rep 1
Reference Control A, rep 2
Reference Control A, rep 3
Co-elution controls Co-elution control for test article
Reference controls Reference Control B, rep 1
Reference Control B, rep 3
First set of replicates Reference Control C, rep 1
Positive Control, rep 1
Test sample, rep 1
Second set of replicates Reference Control C, rep 2
Positive Control, rep 2
Test sample, rep 2
Third set of replicates Reference Control C, rep3
Positive Control, rep 3
Test sample, rep 3
Reference controls Reference Control B, rep 4
Reference Control B, rep 5
Reference Control B, rep 6

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: 1-3

Parameter:

other: Percent Peptide Depletion (PPD) - Lysine

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: The max PPD value across the 3 runs was -0.23, with a mean PPD of 0.00 (negative value) for the 3 runs

Results 2

Run / experiment:

other: 1-3

Parameter:

other: Percent Peptide Depletion (PPD) - Cysteine

Value:

32.12

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: The max PPD value across 3 runs was 3.69, with a mean PPD of 3.05 for the 3 runs.

Any other information on results incl. tables

 

  Cysteine Depletion

The percentage peptide depletion values were as follows:

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
Test Article	11.73	17.28	32.12	34.55	4.06
	11.70		32.29
	10.50		39.24
Positive Control	3.29	17.28	80.96	80.94	0.43
	3.37		80.50
	3.22		81.37

The r²value for the standard calibration curve was 0.99475.

The peptide concentrations for the Reference Controls A and C were as follows:

Reference Control	Peptide Concentration (mM)
	Replicate 1	Replicate 2	Replicate 3	Mean
A	0.46	0.50	0.49	0.48
C	0.48	0.48	0.50	0.49

 

The peak area results for Reference Controls B and C (acetonitrile) were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1	17.38
	2	17.51
	3	18.19
	4	17.02
	5	18.42
	6	16.64
C	1	16.99
	2	16.94
	3	17.91
Mean		17.44
SD		0.61
CV		3.52

Lysine Depletion

The percentage peptide depletion values were as follows:

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
Test Article	25.44	23.51	0.00	0.37	0.64
	24.39		0.00
	23.25		1.11
Positive Control	11.49	23.51	51.13	47.26	4.39
	12.19		48.15
	13.52		42.49

The r²value for the standard calibration curve was 0.99998.

The peptide concentrations for the Reference Controls A and C were as follows:

Reference Control	Peptide Concentration (mM)
	Replicate 1	Replicate 2	Replicate 3	Mean
A	0.50	0.50	0.50	0.50
C	0.52	0.50	0.52	0.51

 

The peak area results for Reference Controls B and C (acetonitrile) were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1	23.81
	2	23.64
	3	23.18
	4	23.70
	5	24.54
	6	23.99
C	1	23.87
	2	22.95
	3	23.69
Mean		23.71
SD		0.46
CV		1.92

 

Applicant's summary and conclusion

Conclusions:

The test article was considered to be negative in the Direct Peptide Reactivity Assay.

Executive summary:

The study was conducted to meet the known requirements of OECD Guidelines for Testing of Chemicals Method 442C (adopted 04 February 2015).

The test article was considered to be negative in the Direct Peptide Reactivity Assay.