3-{2-[2-(3-azaniumylpropoxy)ethoxy]ethoxy}propan-1-aminium di[(2Z)-3-carboxyprop-2-enoate]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 January 2018 to 5 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

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 an in-vitro 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:

3-{2-[2-(3-azaniumylpropoxy)ethoxy]ethoxy}propan-1-aminium di[(2Z)-3-carboxyprop-2-enoate]

EC Number:

818-033-1

Cas Number:

1629579-82-3

Molecular formula:

C18H32N2O11

IUPAC Name:

3-{2-[2-(3-azaniumylpropoxy)ethoxy]ethoxy}propan-1-aminium di[(2Z)-3-carboxyprop-2-enoate]

Test material form:

liquid

Details on test material:

CAS Number: 1629579-82-3
EC Number: 818-033-1
Molecular formula: C18H32N2O11

Specific details on test material used for the study:

Identification: Bis-Aminopropyl Diglycol Dimaleate
Appearance: Clear colourless to pale yellow liquid (determined by Charles River Den Bosch)

Purity/Composition: The test substance is an aqueous solution (74% water as determined in Charles River Project 512944)
Test item storage: At room temperature
Stable under storage conditions until: 30 September 2017 (expiry date)

Additional information
Test Facility Test Item Number: 207396/A
Purity/Composition correction factor: 3.84 (according to water content)
Test item handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Bis-Aminopropyl Diglycol Dimaleate
CAS Number: 1629579-82-3

Molecular formula: C18H32N2O11
Molecular weight: 452.46 g/mol

In chemico test system

Details on the study design:

Objectives
The study was conducted to quantify the reactivity of Bis-Aminopropyl Diglycol Dimaleate 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 the samples were visually inspected for precipitate formation.

Test Item Preparation
A correction factor of 3.84 was used to correct for purity/composition of the test item.
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. 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).
Bis-Aminopropyl Diglycol Dimaleate stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assays 192.11 mg of Bis-Aminopropyl Diglycol Dimaleate was pre-weighed into a clean amber glass vial and dissolved, just before use, in ACN in a total volume of 1528 µL to obtain a 72 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) or lysine (lys) reactivity assay, respectively.

Preparation of Solutions for Cysteine Reactivity Assay
Synthetic Peptide Containing Cysteine (SPCC) Stock Solution
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.

SPCC Reference Control Solutions
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.

SPCC Calibration Curve
A SPCC calibration curve was prepared as described in the table below.

SPCC calibration solutions SPCC concentration (mM) Preparation
STDcys1 0.534 1600 µL stock solution of 0.667 mM SPCC + 400 µL ACN
STDcys2 0.267 1 mL STDcys1 + 1 mL STDcys7
STDcys3 0.134 1 mL STDcys2 + 1 mL STDcys7
STDcys4 0.067 1 mL STDcys3 + 1 mL STDcys7
STDcys5 0.033 1 mL STDcys4 + 1 mL STDcys7
STDcys6 0.017 1 mL STDcys5 + 1 mL STDcys7
STDcys7 0 8 mL phosphate buffer (pH 7.5) + 2 mL ACN

Co-elution Control, Test Item and Positive Control Samples
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in the table below.
Preparation of Co-elution Control, Test Item and Positive Control Samples
Sample Replicates Sample code Preparation
Co-elution control (CC) 1 CCcys-207396/A 750 µL Phosphate buffer pH 7.5, 200 µL ACN, 50 µL 207396/A test solution (100 mM)
Cinnamic aldehyde (PC) 3 PCcys-1 to PCcys-3 750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN, 50 µL Cinnamic aldehyde solution (100 mM in ACN)
Test item 207396/A 3 207396/A-cys-1 to 207396/A-cys-3 750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN, 50 µL 207396/A test solution (72 mM)

Preparation of Solutions for Lysine Reactivity Assay

Synthetic Peptide Containing Lysine (SPCL) Stock Solution
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

SPCL Reference Control Solutions
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.

SPCL Calibration Curve
A SPCL peptide calibration curve was prepared as described in the table below.

Preparation of SPCL Calibration Curve
SPCL calibration solutions SPCL concentration (mM) Preparation
STDlys1 0.534 1600 µL stock solution of 0.667 mM SPCL + 400 µL ACN
STDlys2 0.267 1 mL STDlys1 + 1 mL STDlys7
STDlys3 0.134 1 mL STDlys2 + 1 mL STDlys7
STDlys4 0.067 1 mL STDlys3 + 1 mL STDlys7
STDlys5 0.033 1 mL STDlys4 + 1 mL STDlys7
STDlys6 0.017 1 mL STDlys5 + 1 mL STDlys7
STDlys7 0 8 mL ammonium acetate buffer (pH 10.2) + 2 mL ACN

Co-elution Control, Test Item and Positive Control Samples
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in the table below.

Preparation of Co-elution Control, Test Item and Positive Control Samples
Sample Replicates Sample code Preparation
Co-elution control (CC) 1 CClys-207396/A 750 µL Ammonium acetate buffer pH 10.2, 250 µL 207396/A test solution (100 mM)
Cinnamic aldehyde (PC) 3 PClys-1 to PClys-3 750 µL Stock solution of 0.667 mM SPCL, 250 µL Cinnamic aldehyde solution, (100 mM in ACN)
Test item 207396/A 3 207396/A-lys-1 to 207396/A-lys-3 750 µL Stock solution of 0.667 mM SPCL, 250 µL 207396/A test solution (72 mM)

Sample Incubations
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. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24±2 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.

HPLC-PDA Analysis
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed using the following system:
System 1 (used for Cysteine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
• LC Column oven 300 (Thermo Scientific)
• Surveyor PDA detector (Thermo Scientific) System 2 (used for Lysine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
• Column Oven #151006 (Grace, Worms, Germany)
• Surveyor PDA detector (Thermo Scientific)

All samples were analyzed according to the HPLC-PDA method presented in Table 1 (attached). The HPLC sequences of the cysteine and lysine reactivity assay for BisAminopropyl Diglycol Dimaleate are presented in Table 2 (attached).

Sample Collection and Analysis
No analysis of the formulated test item was conducted for this study with respect to either test item concentration, homogeneity or of the test item in the vehicle. Nevertheless, according to OECD Guidelines relative to the application of the Good Laboratory Practice Principles to short-term studies, the preparations were performed with approved procedures and documented in detail. Preparations were visually homogenous prior to use and all preparations were used within 4 hours after adding the vehicle to the test item. This GLP exception was therefore considered as being minor with no impact on the outcomes and the integrity and the achievement of the objective of the study.

Test System
Test system Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.
Rationale Recommended test system in the international OECD guideline for DPRA studies.
Source JPT Peptide Technologies GmbH, Berlin, Germany.
Batch See report for detailed information.
Storage The peptides were stored in the freezer (≤-15°C) for a maximum of 6 months.

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: 5 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

Positive control results:

Cysteine Reactivity
The results of the Reference Control samples A and C are presented in Table 4 (attached). The mean peptide concentration of Reference Controls A was 0.512±0.007 mM while the mean peptide concentration of Reference Controls C was 0.509±0.018 mM. The mean Reference Control samples A and C were both within the acceptance criteria of 0.50±0.05 mM.

Lysine Reactivity Assay
The results of the Reference Control samples A and C are presented in Table 10 (attached). The mean peptide concentration of Reference Controls A was 0.499±0.009 mM while the mean peptide concentration of Reference Controls C was 0.478±0.016 mM. The mean Reference Control samples A and C were both within the acceptance criteria of 0.50±0.05 mM.

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: %SPCC depletion

Remarks:

Mean

Value:

92.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: %

Results 2

Key result

Parameter:

other: %SPCL depletion

Remarks:

Mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: %

Results 3

Key result

Parameter:

other: Mean %SPCC/SPCL depletion

Value:

46.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: %

Other effects / acceptance of results:

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the 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 Bis-Aminopropyl Diglycol Dimaleate, were all within the acceptability criteria for the DPRA.

Any other information on results incl. tables

Solubility Assessment of Bis-Aminopropyl Diglycol Dimaleate

At a concentration of 72 mM, Bis-Aminopropyl Diglycol Dimaleate was soluble in ACN and therefore this solvent was used to dissolve Bis-Aminopropyl Diglycol Dimaleate in this DPRA study.

Solubility Assessment of Bis-Aminopropyl Diglycol Dimaleate

At a concentration of 72 mM, Bis-Aminopropyl Diglycol Dimaleate was soluble in ACN and therefore this solvent was used to dissolve Bis-Aminopropyl Diglycol Dimaleate in this DPRA study.

Cysteine Reactivity Assay

The reactivity of Bis-Aminopropyl Diglycol Dimaleate towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC-PDA analysis, following 25 hours of incubation at 25 ± 2.5°C. Representative chromatograms of CCcys-207396/A and 207396/A-cys samples are presented in the attached Appendix. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 3 (attached).

Acceptability of the Cysteine Reactivity Assay

The SPCC standard calibration curve is presented in Figure 1 (attached). The correlation coefficient (r2) of the SPCC standard calibration curve was 0.993. Since the r2was >0.99, the SPCC standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented in Table 4 (attached). The mean peptide concentration of Reference Controls A was 0.512 ± 0.007 mM while the mean peptide concentration of Reference Controls C was 0.509 ± 0.018 mM. The mean Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve Bis-Aminopropyl Diglycol Dimaleate did not impact the Percent SPCC Depletion.

The SPCC peak areas for Reference controls B and C are presented in Table 5 (attached). The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 2.4%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCC A220/A258area ratios of Reference controls A, B and C are presented in Table 6 (attached). The mean area ratio (A220/A258) of the Reference Control samples was 17.96. The mean A220/A258 ratio ± 10% range was 16.16 - 19.76. Each sample showing an A220/A258ratio within this range gives an indication that co-elution has not occurred.

The results of the positive control cinnamic aldehyde are presented in Table 7 (attached). The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 72.8% ± 1.0%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Results Cysteine Reactivity Assay for Bis-Aminopropyl Diglycol Dimaleate

Preparation of a 72 mM Bis-Aminopropyl Diglycol Dimaleate stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after approximately 24 hours of incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. No precipitate was observed in any of the samples. 

The results of the cysteine reactivity assay for Bis-Aminopropyl Diglycol Dimaleate are presented in Table 8 (attached). In the CC sample no peak was observed at the retention time of SPCC (see chromatogram in the attached Appendix). This demonstrated that there was no coelution of the test item with SPCC. For the 207396/A-cys samples, the mean SPCC A220/A258area ratio was 30.52. This was outside the 16.16-19.76 range. However, since the test item displayed high reactivity towards SPCC, accurate calculation of the peak purity was not possible due to the low SPCC signal at 258 nm. Overall, it can be concluded that the test item did not co-elute with SPCC.

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for Bis-Aminopropyl Diglycol Dimaleate was 92.3% ± 1.4%.

Lysine Reactivity Assay

The reactivity of Bis-Aminopropyl Diglycol Dimaleate towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis, following 25 hours of incubation at 25±2.5°C. Representative chromatograms of CClys-207396/A and 207396/A-lys samples are presented in the attached Appendix. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 9 (attached).

Acceptability of the Lysine Reactivity Assay

The SPCL standard calibration curve is presented in Figure 2 (attached). The correlation coefficient (r2) of the SPCL standard calibration curve was 0.993. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented in Table 10 (attached). The mean peptide concentration of Reference Controls A was 0.499 ± 0.009 mM while the mean peptide concentration of Reference Controls C was 0.478 ± 0.016 mM. The mean Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve Bis-Aminopropyl Diglycol Dimaleate did not impact the Percent SPCL Depletion.

The SPCL peak areas for Reference controls B and C are presented in Table 11 (attached). The CV of the peptide areas for the nine Reference Controls B and C was 2.7%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCC A220/A258area ratios of Reference controls A, B and C are presented in Table 12 (Appendix 4). The mean area ratio (A220/A258) of the Reference Control samples was 13.99. The mean A220/A258ratio ± 10% range was 12.59-15.39. Each sample showing an A220/A258ratio within this range gives an indication that co-elution has not occurred.

The results of the positive control cinnamic aldehyde are presented in Table 13 (attached). The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 60.8% ± 0.9%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Results Lysine Reactivity Assay for Bis-Aminopropyl Diglycol Dimaleate

Preparation of a 72 mM Bis-Aminopropyl Diglycol Dimaleate stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after approximately 24 hours of incubation, both the CC as well as the test item samples were visually inspected. No precipitate was observed in any of the samples.  

The results of the lysine reactivity assay for Bis-Aminopropyl Diglycol Dimaleate are presented in Table 14 (attached). In the CC sample no significant peak was observed at the retention time of SPCL (see chromatogram in the attached Appendix). This demonstrated that there was no co-elution of the test item with SPCL. For the 207396/A-lys samples, the mean SPCL A220/A258area ratio was 13.94. Since this was within the 12.59 - 15.39 range, this again indicated that there was no co-elution of the test item with SPCL.

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for Bis-Aminopropyl Diglycol Dimaleate was 0.0% ± 0.0%.

Reactivity Classification

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 92.3% SPCC depletion while in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 46.2% and as a result the test item was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Therefore, Bis-Aminopropyl Diglycol Dimaleate was considered to be positive in the DPRA.

SPCC and SPCL Depletion and Reactivity Classification for Bis-Aminopropyl Diglycol Dimaleate 

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	Reactivity class
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Bis- Aminopropyl Diglycol Dimaleate	92.3%	±1.4%	0.0%	±0.0%	46.2%	High reactivity

SD = Standard Deviation

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

See RSS endpoint: Skin sensitisation, WoE, Charles River (2020); Weight of Evidence

Conclusions:

In conclusion, the OECD 442C DPRA test is valid. Bis-Aminopropyl Diglycol Dimaleate was positive in the DPRA assay and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The objective of this DPRA (OECD 442C) study was to determine the reactivity of Bis-Aminopropyl Diglycol Dimaleate towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL).

After incubation of Bis-Aminopropyl Diglycol Dimaleate 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 which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

The study procedures described in this report were based on the most recent OECD guidelines (OECD 442C).

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve Bis-Aminopropyl Diglycol Dimaleate and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. An overview of the obtained assay validation parameters is presented in the table below:

Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.993	>0.99	0.993
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.512 ± 0.007	0.50 ± 0.05	0.499 ± 0.009
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.509 ± 0.018	0.50 ± 0.05	0.478 ± 0.016
CV (%) for RC samples B and C	<15.0	2.4	<15.0	2.7
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	72.8	40.2-69.0	60.8
SD of peptide depletion cinnamic aldehyde (%)	<14.9	1.0	<11.6	0.9
SD of peptide depletion for   Bis-Aminopropyl Diglycol Dimaleate (%)	<14.9	1.4	<11.6	0.0

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation; NA = Not Applicable.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the 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 Bis-Aminopropyl Diglycol Dimaleate, were all within the acceptability criteria for the DPRA.  No co-elution of the test item with SPCC or SPCL was observed. 

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 92.3% SPCC depletion while in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 46.2% and as a result Bis-Aminopropyl Diglycol Dimaleate was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

SPCC and SPCL Depletion and Reactivity Classification for Bis-Aminopropyl Diglycol Dimaleate

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	Reactivity class
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Bis- Aminopropyl Diglycol Dimaleate	92.3%	±1.4%	0.0%	±0.0%	46.2%	High reactivity

SD = Standard Deviation

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. Bis-Aminopropyl Diglycol Dimaleate was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

AMMENDMENT 1:

Since the correction factor that was used to prepare the test item stock solution in CAN was calculated incorrectly, a correction factor of 2.778 was used where a correction factor of 3.84 should have been used. As a result, the prepared test item stock solution had a concentration of 72 mM instead of the desired 100 mM and the SPCC and SPCL incubations were performed at lower concentrations than intended. However, since at this lower concentration the test item was already classified in the highest reactivity class, this calculation error had no impact on the outcome of the study.