Dioctyl maleate, branched

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 October 2017- 23 November 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Results were very weakly positive with low reactivity

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Regulation 1223/2009, Article 18, restricts the use of in vivo studies on cosmetic raw materials (the sole use of this substance), therefore a recognised in chemico test was conducted on the test material.

Test material

Test material information

Constituent 1

Reference substance name:

Dioctyl maleate, branched

EC Number:

287-673-6

EC Name:

Dioctyl maleate, branched

Cas Number:

85566-63-8

Molecular formula:

C20H36O4

IUPAC Name:

1,4-bis(octan-2-yl) (2Z)-but-2-enedioate

Test material form:

liquid

Specific details on test material used for the study:

Test Article Bernel Ester DCM
CAS Number 85566-63-8
Storage 15 to 25˚C, protected from light
Purity 92.78%*

* Assumed 100% for testing

In chemico test system

Details on the study design:

Test Article Formulation
The test article was dissolved in acetonitrile. This was the first of the listed vehicles that produced a visually clear solution at a concentration of 100 mM.
The positive control was dissolved in acetonitrile at a concentration of 100 mM.
A stock solution containing cysteine at approximately 0.667 mM was prepared in 100 mM Phosphate Buffer pH 7.5 and a stock solution containing lysine at approximately 0.667 mM was prepared in 100 mM ammonium acetate buffer pH 10.2.
Formulations were prepared shortly before testing.

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 for 24±2 hours. At the end of the incubation period the samples were visually inspected for precipitate formation.

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 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).
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 2
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

DATA EVALUATION
Data Analysis and Calculations
The chromatographic data were collected and processed using Chromeleon, a validated data capture system.
A calibration curve was generated based on the concentration of standards and the peak area. All the peaks of the samples were integrated (standards, samples and controls) “valley to valley” (when possible) and Cysteine and Lysine peptides concentrations were determined from absorbance at 220 nm using the respective calibration curves. Areas were determined for all the samples.
The PPD of each peptide was determined in each sample with the following formula:

PPD = (1-(Peptide peak area in replicate injection/Mean peptide peak area in reference controls C) x 100

The mean peptide peak areas for the nine Reference Controls B and C, Standard Deviation (SD) and Coefficient of Variation (CV) were calculated.
The mean peptide peak areas and the mean peptide concentrations (mM) for the three Reference Controls C were calculated.
The chromatograms of Co-elution Controls were compared to the chromatograms of Reference Controls C.
For the positive control and for the test article, the PPD in each replicate was calculated from the peptide area of the replicate injection and the mean peptide peak for Reference Control C. The PPD of every injected positive control and test chemical replicate was calculated. The mean PPD of the three replicate determinations and SD were calculated.
The mean percent cysteine and percent lysine depletion values were calculated for each test chemical (negative depletion is considered as “0” when calculating the mean).

Assay Acceptance Criteria
The following criteria should be met for a run to be considered valid:
• The standard calibration curve should have a r2>0.99.
• The mean peptide concentration for reference controls A should be 0.50±0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C should be <15.0%.
• The mean PPD value of the three replicates for the positive control and maximum standard deviation (SD) must fall within the ranges in the following table:

Peptide Mean PPD Values (%)
Lower Bound Upper Bound SD
Cysteine 60.8 100 <14.9
Lysine 40.2 69.0 <11.6

The following criteria should be met for the test article’s result to be considered valid:
• The maximum standard deviation for the test article replicates should be <14.9 for the percent cysteine depletion and <11.6 for the percent lysine depletion.
• The mean peptide concentration of the three reference controls C in the appropriate should be 0.50±0.05 mM.

#Prediction Model
The mean percent cysteine and percent lysine depletion value was calculated. By using the cysteine 1:10/lysine 1:50 prediction model below, the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an IATA.

Mean of Cysteine and Lysine % Depletion Reactivity Class DPRA Prediction
0% ≤ mean % depletion ≤6.38% No or minimal reactivity Negative
6.38% < mean % depletion ≤22.62% Low reactivity
22.62% < mean % depletion ≤42.47% Moderate reactivity Positive
42.47% < mean % depletion ≤100% High reactivity

The mean percent depletion fell in the range of 3% to 10% for the cysteine 1:10/lysine 1:50 prediction model, therefore a second run was required.

Results and discussion

Positive control results:

Cinnamic aldehyde (CAS No. 104-55-2, batch number MKBT8955V purity 99.1%, expiry 29 February 2020) was used as the positive control.
The mean percentage peptide depletion (PPD) values for the positive control were:
- 62.06for lysine
- 51.04 for cysteine

In vitro / in chemico

Results

Results 1

Run / experiment:

other: 1-3

Parameter:

other: % peptide depletion (PPD) lysine

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: 1-3

Parameter:

other: % peptide depletion (PPD) cysteine

Value:

6.29

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

All acceptance criteria were met in both Experiments, with the exception that in Experiment 2, the mean peptide concentration for Reference Control A was just below the range specified, at 0.43 mM and 0.44 mM for cysteine and lysine, respectively. As the routine system suitability test prior to sample analysis was satisfactory, the results were considered valid.

Any other information on results incl. tables

Lysine Depletion, Experiment 1

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	33.86	38.04	10.99	8.02	2.68
	35.84		5.78
	35.27		7.28
Positive Control	13.16	38.04	65.4	62.06	3.41
	15.75		58.6
	14.39		62.17

The r² value for the standard calibration curve was 0.99995.

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	0.47	0.48	0.47
C	0.47	0.48	0.48	0.48

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

Reference Control	Replicate	Peptide Peak Area
B	1	37.49
	2	36.67
	3	40.15
	4	38.58
	5	39.19
	6	39.42
C	1	37.28
	2	38.43
	3	38.41
Mean		38.4
SD		1.11
CV		2.89

Lysine Depletion, Experiment 2

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	33.85	28.84	0	1.24	1.25
	28.12		2.5
	28.49		1.21
Positive Control	13.75	28.84	52.32	51.04	9.89
	17.14		40.57
	11.47		60.23

The r² value for the standard calibration curve was 0.99994.

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	0.44	0.43	0.44	0.44
C	0.45	0.45	0.46	0.45

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

Reference Control	Replicate	Peptide Peak Area
B	1	33.42
	2	27.95
	3	27.71
	4	28.5
	5	31.77
	6	28.03
C	1	28.72
	2	28.37
	3	29.45
Mean		29.32
SD		1.97
CV		6.7

Cysteine Depletion, Experiment 1

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	24.75	26.41	6.29	7.03	0.65
	24.45		7.42
	24.46		7.38
Positive Control	6.34	26.41	75.99	76.47	1.46
	6.52		75.31
	5.78		78.11

The r² value for the standard calibration curve was 0.99865.

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	0.44	0.49	0.5	0.48
C	0.49	0.48	0.49	0.49

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

Reference Control	Replicate	Peptide Peak Area
B	1	26.53
	2	26.61
	3	27.12
	4	25.89
	5	25.58
	6	25.94
C	1	26.86
	2	25.87
	3	26.48
Mean		26.32
SD		0.52
CV		1.98

Cysteine Depletion, Experiment 2

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	15.46	17.2	10.12	12.29	7.17
	13.71		20.29
	16.09		6.45
Positive Control	6.11	17.2	64.48	65.72	4.66
	6.57		61.8
	5.01		70.87

The r² value for the standard calibration curve was 0.99874.

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	0.41	0.44	0.42	0.43
C	0.44	0.46	0.44	0.45

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

Reference Control	Replicate	Peptide Peak Area
B	1	16.55
	2	16.9
	3	15.05
	4	14.04
	5	14.16
	6	14.89
C	1	17.13
	2	17.7
	3	16.78
Mean		15.91
SD		1.38
CV		8.66

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The test article, Bernel Ester DCM, was considered to be weakly positive (low reactivity) in the Direct Peptide Reactivity Assay.

Executive summary:

The study was conducted to quantify the reactivity of Bernel Ester DCM towards model synthetic peptides containing either lysine or cysteine. The data may be 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 test article was dissolved in acetonitrileat a concentration of 100 mM.

The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24±2 hours in glass autosampler vials, protected from light and set at 25°C.

The remaining concentration of cysteine- or lysine-containing peptides following the 24 hour incubation period was measured by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

In Experiment 1, the cysteine depletion value was 7.03%, the lysine depletion value was 8.02% and the mean of the cysteine and lysine depletion values was 7.52%. As the results fell within the range of 3% to 10% for the cysteine 1:10/lysine 1:50 prediction model, a further run was conducted.

In Experiment 2, the cysteine depletion value was 12.29%, the lysine depletion value was 1.24% and the mean of the cysteine and lysine depletion values was 6.76%. These results were concordant with those of Experiment 1.

All acceptance criteria were met in both Experiments, with the exception that in Experiment 2, the mean peptide concentration for Reference Control A was just below the range specified, at 0.43 mM and 0.44 mM for cysteine and lysine, respectively. As the routine system suitability test prior to sample analysis was satisfactory, the results were considered valid.

The test article, Bernel Ester DCM, was considered to be weakly positive with low reactivity in the Direct Peptide Reactivity Assay.