Registration Dossier

Administrative data

Description of key information

Skin Sensitisation


 


Skin sensitisation, Key Study, in-vitro, Charles River (2017 and 2018); KeratinoSens; OECD 442D


2018 Study


Bis-Aminopropyl Diglycol Dimaleate showed no toxicity (no IC30 and IC50 value) in the current experiment. This result is similar to the results that were observed in the two experiments performed in Test Facility Study No.518597.


A biologically relevant, dose-related induction of the luciferase activity (EC1.5value 269 µM) with a maximum induction of 2.36-fold was measured in the current experiment. In Test Facility Study No. 518597 two KeratinoSens runs were performed. One assay run gave a positive result (EC1.5of 757 µM) and the second assay run gave a negative result (EC1.5of 1427 µM but no induction below 1000 µM).


 


Bis-Aminopropyl Diglycol Dimaleate is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed in 2 out of 3 experiments at test concentrations ≤ 1000 µMwith a cell viability >70%.


In conclusion, based on the current study and the two experiments performed in Test Facility Study No. 518597, Bis-Aminopropyl Diglycol Dimaleate is classified as positive (activation of the antioxidant / electrophile responsive element (ARE)-dependent pathway in keratinocytes) in the KeratinoSensTM assay.


 


2017 Study


The test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (EC 1.5 values of 1048 µM and 1975 µM in experiment 1 and 2, respectively) was measured at concentrations lower than 1000 µM in both experiments. The maximum luciferase activity induction (Imax) was 1.76-fold and 1.51-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSensTM assay since in both experiments no biological relevant luminescence induction at concentrations below 1000 µM was observed.


 


In conclusion, Bis-Aminopropyl Diglycol Dimaleate is classified as negative (no biological relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.


 


Skin sensitisation, Key Study, Charles River (2018); DPRA; OECD 442C


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.


 


Skin sensitisation, Key Study, Charles River (2018); USENS: OECD 442E


In conclusion, Bis-Aminopropyl Diglycol Dimaleate is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.


 


Skin sensitisation, Key Study, Charles River (2018); DEREK


DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. Bis-Aminopropyl Diglycol Dimaleate is predicted to be not sensitizing to the skin.


 


Skin sensitisation, WoE, Charles River (2020); Weight of Evidence


Based on the in chemico/in vitro results and human data for bis-aminopropyl diglycol dimaleate and available data from public literature for maleic acid on skin sensitizing properties, it cannot be excluded that bis-aminopropyl diglycol dimaleate possess skin sensitization properties.


The negative HRIPT (2014) data are not considered sufficient evidence that formulations containing bis-aminopropyl diglycol dimaleate up to and including 20% do not warrant classification and labelling of these products for skin sensitization due to the small number of subjects (50 to 67 while normally at least 100 is required). More information on the sensitizing potency of the formulation(s) is needed to demonstrate non skin sensitizing properties for the formulation(s).


 


Skin sensitisation, WoE, QACS Lab (2021) and Bioscreen (2020); HRIPT


These studies are reported in section 7.10.4 of the IUCLID dossier.


QACS Lab (2021) - Throughout the study, the number of volunteers that presented an irritant reaction was six percent (6%). The number of volunteers that presented an allergic reaction was zero percent (0%). According to the experimental conditions of the study, the test product, can be considered as "Hypoallergenic”. 100 healthy adult volunteers completed the study.


BioScreen (2020) - No adverse reactions of any kind were reported during the course of this study. Under the conditions of the study, there was no indication of a potential to elicit dermal irritation or sensitization (contact allergy) noted. 104 healty adult volunteers completed the study.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
21 March 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
The result as generated by DEREK NEXUS is presented in Appendix 2 (attached). The relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) are presented in Appendix 3 (attached).
Qualifier:
according to guideline
Guideline:
other: DEREK NEXUS
Version / remarks:
The objective of this study was to obtain a prediction on the potential for skin sensitization of Bis-Aminopropyl Diglycol Dimaleate with the in silico model DEREK NEXUS. In this assessment version 5.0.2 of DEREK NEXUS was used.
Principles of method if other than guideline:
DEREK NEXUS is a knowledge-based system that contains 80 alerts for skin sensitization based on the presence of molecular substructures. LHASA (see Appendix I) has inserted validation comments for the skin sensitization alerts.
GLP compliance:
no
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.
Specific details on test material used for the study:
- Chemical name: Bis-Aminopropyl Diglycol Dimaleate
- CAS Number: 1629579-82-3
- Molecular weight: 452.46
- Molecular formula: C18H32N2O11
Key result
Run / experiment:
other: DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. Bis-Aminopropyl Diglycol Dimaleate is predicted to be not sensitizing to the skin.
Remarks on result:
no indication of skin sensitisation

The result as generated by DEREK NEXUS is presented in Appendix 2 (attached).

The relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) are presented in Appendix 3 (attached).

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. Bis-Aminopropyl Diglycol Dimaleate is predicted to be not sensitizing to the skin.

Interpretation of results:
other: Bis-Aminopropyl Diglycol Dimaleate is predicted to be not sensitizing to the skin.
Remarks:
See RSS endpoint: Skin sensitisation, WoE, Charles River (2020); Weight of Evidence
Conclusions:
The result as generated by DEREK NEXUS is presented in Appendix 2. The relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) are presented in Appendix 3.
DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. Bis-Aminopropyl Diglycol Dimaleate is predicted to be not sensitizing to the skin.
Executive summary:

Introduction

The objective of this study was to obtain a prediction on the potential for skin sensitization of Bis-Aminopropyl Diglycol Dimaleate with the in silico model DEREK NEXUS. In this assessment version 5.0.2 of DEREK NEXUS was used.

 

Background/scope

DEREK NEXUS is a knowledge-based system that contains 80 alerts for skin sensitization based on the presence of molecular substructures. LHASA (see Appendix I) has inserted validation comments for the skin sensitization alerts.

 

The level of likelihood of a structure being sensitizing to skin is expressed in terms of:

- Certain: There is proof that the proposition is true.

- Probable: There is at least one strong argument that the proposition is true and there are no arguments against it.

- Plausible: The weight of evidence supports the proposition.

- Equivocal: There is an equal weight of evidence for and against the proposition.

 

The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which are evaluated as being equivocal or greater was used in this assessment.

 

If a substance is predicted to be a skin sensitizer, its potency is predicted by DEREK NEXUS by calculating an EC3value based on experimental data from the closest structurally-related substances (at least 3 substances should be present) using the following equation:

 

EC3Q = MWQ /(Σ ωNN / Σ TNN)

 

MW = molecular weight

T = Tanimoto similarity score

ω = weighting factor = (MWNN/EC3) * TNN

Q = query compound

NN = nearest neighbour

 

The EC3 is the estimated concentration needed to produce a stimulation index of 3.

Conclusion 

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. Bis-Aminopropyl Diglycol Dimaleate is predicted to be not sensitizing to the skin.

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
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.
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

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

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.
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: %
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: %
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.

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

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 (r2) 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.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
28 Nov 2017 to 02 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U-SENS™)’ (9 October 2017).
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
U937 cell line activation test (U-SENS™)
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.
Specific details on test material used for the study:
Identification: Bis-Aminopropyl Diglycol Dimaleate
Appearance: Clear to transparent yellow liquid
Purity/Composition: Not indicated
Test item storage: At room temperature

Additional information
Test facility test item number: 207396/C
Purity/composition correction factor: Contains 74% water
Test item handling: No specific handling conditions required
Stability at higher temperatures: Yes, maximum temperature: 40°C, maximum duration: 60 minutes
Chemical name (IUPAC), synonym or trade name: Bis-Aminopropyl Diglycol Dimaleate
CAS Number: 1629579-82-3
Molecular formula: C18H32N2O11
Molecular weight: 452.46
Solubility in vehicle: Complete medium: Not indicated
Stability in vehicle: Complete medium: Not indicated
Details of test system:
U-937 cell line [442E]
Details on the study design:
The objective of this study is to evaluate the ability of Bis-Aminopropyl Diglycol Dimaleate to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay.
Background of the test system
The existing knowledge of the chemical and biological mechanisms associated with skin sensitization has been summarized in the form of an Adverse Outcome Pathway (AOP). The third key event in this AOP is the activation of dendritic cells (DC), typically assessed by expression of specific cell surface markers, chemokines and cytokines.
The U-SENS™ method is proposed to address this third key event by quantifying the change in the expression of a cell surface marker associated with the process of activation of monocytes and DC (i.e. CD86), in the human histiocytic lymphoma cell line U937, following exposure to sensitisers. The measured expression levels of CD86 cell surface marker in the cell line U937 is then used for supporting the discrimination between skin sensitisers and non-sensitisers.

Vehicle
The vehicle of the test item, i.e. complete medium (RPMI-1640, Life Technologies, Bleiswijk, The Netherlands).

Negative Control
Lactic Acid (LA, RS471) is used as negative control. On the treatment day, a solution at 10 mg/mL was prepared in RPMI medium. This solution was diluted 1:25 in order to obtain a 0.4 mg/mL stock solution (final dose level 200 µg/mL).
Identification: DL-Lactic Acid
CAS Number: 50-21-5
Molecular formula: CH3CH(OH)COOH
Molecular weight: 90.08
Appearance: Colourless viscous liquid
Batch: BCBK8387V
Purity: 90.7%
Storage conditions: At room temperature
Stable under storage conditions until: 24 July 2018

Positive Control
2,4,6-Trinitrobenzenesulfonic acid (TNBS; RS599) was provided as 1 M solution. On the treatment day a 10 mg/mL solution was prepared. This solution was diluted 1:100 in order to obtain a 0.1 mg/mL stock solution (final dose level 50 µg/mL).
Identification: PICRYLSULFONIC ACID SOLUTION, 1M in H2O
CAS Number: 2508-19-2
Molecular formula: C6H3N3O9S
Molecular weight: 293.17
Appearance: Dark yellow liquid (determined by Charles River Den Bosch)
Batch: BCBR6376V
Purity: Not indicated
Storage conditions: In freezer (-15°C)
Stable under storage conditions until: 31 July 2018 (retest date)

Dose Formulation and Analysis

Preparation of Test Item Stock, Spiking and Working Solutions

A correction factor of 3.85 was made for the water content of the test item (74% water).
A solubility test was performed. The test item was either dissolved or suspended in complete medium and DMSO to a final concentration of 50 mg/mL. The test item formed a clear solution in complete medium at 50 mg/mL (yellow solution). In DMSO the test item formed clear solution at 50 mg/mL (colourless solution). Complete medium was used as solvent for the main experiments.

In the main experiments the test item was dissolved in complete medium at 0.4 mg/mL (yellow). The stock was diluted to a final test concentrations of 200, 100, 50, 20, 10 and 1 µg/mL and 200, 100, 50, 10, 5 and 1 µg/mL in experiment 1 and 2, respectively, in the 96-well plate.
No precipitation was observed at the end of the incubation period in the 96-well plates.
Test item concentrations were used within 2 hours after preparation.
Any residual volumes were discarded.

Test System

Test System: U937 human monocytes.
Justification: Inducible CD86-expressing cells
Source: ATCC (American Type Culture Collection, Virginia, USA).
ATCC no.: CRL-1593.2TM.

Stock cultures of these cells are stored in liquid nitrogen (-196°C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. Once a year the cell line is checked for infection with a mycoplasma detection test.
Each batch of cells received from a supplier are submitted to a qualification process to guarantee their suitability (spontaneous CD86 level) for the test by comparison with the historical data or data from the literature.

Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended for skin sensitization to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the U-SENSTM assay, which is recommended in international guideline (e.g. OECD).

Cell Culture
Cell culture medium
Stock and treatment cultures were performed in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (FCS), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively).

Environmental conditions
All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 85 –
99 %) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.1 – 36.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Plating of Cells
Cultures were initiated in 96-well plates using 100 µL/well of a cell suspension adjusted at 5.0 x 105 viable cells/mL. If the cell viability is < 90% the cells were not used. All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of complete medium untreated control, solvent/vehicle control, negative and positive controls were tested.

Number of experiments
Two experiments were conducted per test item to demonstrate reproducibility of the results and conclusion.

Treatment of Cells
Cells are treated for 45 ± 3 hours with the selected doses. The test item was in the first experiment evaluated up to 200 µg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 µg/mL. A negative untreated control (culture medium), a vehicle control and the positive and negative control items were included. The final volume in the wells was 200 µL.

In the second experiment and following, if applicable, cells were treated with six selected doses of test item. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second experiment were 1.0, 5, 10, 50, 100 and 200 µg/mL.

Precipitate evaluation
After 45 ± 3 hours of exposure, wells were checked for precipitate. If any, precipitate is documented in the raw data and reported in the table of the results.

Cell antibodies staining for IgG1 and CD86
Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded and cells were rinsed once with Phosphate Buffered Saline (PBS) containing 5% FCS. After a second centrifugation step 100 µL/well of staining buffer (PBS containing 5% FCS) was applied to the cells.
FITC-conjugated antibodies was used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control (#555748; BD, Amsterdam, The Netherlands)
- Human CD86 specific mouse IgG1 (#555657; BD, Amsterdam, The Netherlands)
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 µL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 µL of PBS.

Flow cytometry method

Acquisition
Just before acquisition, 5 µL of a 0.5 µg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. For the acquisition the BD FACSCanto™ flow cytometer was used and for further analysis BD FACSDiva™ software was used.

Analysis
All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, for the analysis of all the other wells. The P1 region was adjusted if necessary in a SSC (X-axis) and FSC (Y-axis) plot.
The P2 region was defined for the PI negative cells among P1 in a histogram with counts (Y-axis) and PI fluorescence (X-axis). The amount of cytotoxicity were analyzed as percentage of cells in P2. The P2 region was then plotted in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant was placed according acceptability criterion b. The percentage of cells in the UR quadrant was used to calculate the stimulation index.

Color Interferences
On IgG1 analysis
There is colour interference in the IgG1 evaluation when the X Median of the FITC-fluorescence in the UL Quad is 50% higher than the X Median fluorescence of the vehicle control IgG1 well (IgG1 X Median S.I. ≥ 150%).

ACCEPTABILITY CRITERIA
• At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells is > 90%
• When DMSO is used as a solvent, the validity of the DMSO vehicle control is assessed by calculating a DMSO S.I. compared to untreated cells, and the mean viability of the triplicate cells is > 90%. The DMSO vehicle control is valid if the mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells.
• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25%.
• At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5%.
• No drift in CD86 expression is observed. A drift is defined by i) the corrected %CD86+ value of the untreated control replicate 3 is less than 50% of the mean of the corrected %CD86+ value of untreated control replicates 1 and 2; and ii) the corrected %CD86+ value of the negative control replicate 3 is less than 50% of mean of the corrected %CD86+ value of negative control replicates 1 and 2.
• The positive control (TNBS) is considered as valid if at least two out of the three wells are positive (CD86 S.I. ≥ 150%) and non-cytotoxic (cell viability ≥ 70%).
• Negative control LA is considered as valid if at least 2 out of 3 LA wells are negative (CD86-IgG1 SI < 150) and non-cytotoxic (cell viability ≥ 70%).

If (one of) the acceptability criteria are not met and the Study Director decides that this has a critical effect on the study, the test was rejected and repeated.

CV70 calculations
When the CV70 cannot be calculated it is indicated as “NA” (Not Applicable). Otherwise the calculation is done as follows:

For each culture (IgG1 well and CD86 well), the percentage of viable cells (PI negative cells) was evaluated. The viability for each dose level is the mean of the IgG1 well and CD86 well.
The theoretical concentration at which the chemical induces 30% cytotoxicity (i.e., 70% viability) was calculated using the following formula:

CV70 = C1 + [(V1 - 70) / (V1 - V2) x (C2 - C1)]

Where:
V1 = the first percentage of viability above 70%
V2= the first percentage of viability below 70%
C1 = dose level corresponding to V1
C2 = dose level corresponding to V2

EC150 calculations
When the EC150 cannot be calculated it is indicated as “NA” (Not Applicable). Otherwise the calculation were done as follows:

For each CD86 well culture, the percentage of induced CD86+ cells is calculated as:
[absolute %CD86+ — absolute%IgG1+]

A stimulation index (S.I.) is calculated for each dose level as follows:
S.I. = ([%CD86+ - %IgG1+] in the treated culture) / (Mean [%CD86+ - %IgG1+] of the vehicle cultures) x 100

The viability for each dose level are the mean of the IgG1 well and CD86 well.
The theoretical concentration at which the chemical induces a S.I. of 150 (i.e., 50% of CD86+ cells over the vehicle control) was calculated using the following formula:
EC150 = C1 + [(150 – S.I. 1) / (S.I. 2 – S.I. 1)] x (C2 – C1)

Where:
S.I.1 = the first percentage of CD86+ below 150%
S.I.2 = the first percentage of CD86+ above 150%
C1 = dose level corresponding to S.I. 1
C2 = dose level corresponding to S.I. 2

INTERPRETATION
• For CD86 expression measurement, each test chemical is tested in at least two independent runs (performed on a different day) to derive a single prediction (NEGATIVE or POSITIVE).
• The individual conclusion of an U-SENS™ run is considered Negative (hereinafter referred to as N) if the S.I. of CD86 is less than 150% at all non-cytotoxic concentrations (cell viability ≥ 70%) and if no interference (cytotoxicity, solubility or colour) is observed. Solubility interference is defined as crystals or drops observed under the microscope at 45 ± 3h post treatment (before the cell staining). Colour interference is defined as a shift of the FITC-labelled IgG1 dot-plot (IgG1 FL1 Geo Mean S.I. ≥ 150%).
• In all other cases: S.I. of CD86 higher or equal to 150% and/or interferences observed, the individual conclusion of an U-SENS™ run is considered Positive (hereinafter referred to as P).
• An U-SENS™ prediction is considered NEGATIVE if at least two independent runs are negative (N) (Figure 1, attached). If the first two runs are both negative (N), the U-SENS™ prediction is considered NEGATIVE and a third run does not need to be conducted.
• An U-SENS™ prediction is considered POSITIVE if at least two independent runs are positive (P) (Figure 1, attached). If the first two runs are both positive (P), the U-SENS™ prediction is considered POSITIVE and a third run does not need to be conducted.
• There is an exception if, in the first run, the S.I. of CD86 is higher or equal to 150% at the highest non-cytotoxic concentration only. The run is then concluded NO CONCLUSION (NC), and additional concentrations (between the highest non cytotoxicity concentration and the lowest cytotoxicity concentration) should be tested in additional runs. A run NC conducts automatically to the need of at least 2 more runs, and to a fourth run in case runs 2 and 3 are not concordant (N and/or P independently) (Figure 1, attached). Follow up runs will be considered positive even if only one non cytotoxic concentration gives a CD86 equal or above 150%, since the dose setting has been adjusted for the specific test chemical. The final prediction will be based on the majority result of the three or four individual runs (i.e. 2 out of 3 or 2 out of 4) (Figure 1, attached).


Vehicle / solvent control:
cell culture medium
Negative control:
DL-Lactic acid
Positive control:
picrylsulfonic acid/2,4,6-trinitro-benzene-sulfonic acid (TNBS) [442E]
Positive control results:
The positive control (TNBS) showed a S.I. ≥ 305% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The test was considered valid.
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
EC150, CD86 [442E]
Value:
9.5 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
CV70 [442E]
Value:
182 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
EC150, CD86 [442E]
Value:
2.8 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Both tests passed the acceptance criteria:

• At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (99% in experiment 1 and 98% in experiment 2).
• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in both experiments.
• At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5% in both experiments.
• No drift in CD86 expression was observed in the untreated controls and negative controls.

In both experiments the positive and negative control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The objective of this study is to evaluate the ability of Bis-Aminopropyl Diglycol Dimaleate to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay.

 

The test item was evaluated for the ability to increase the expression levels of CD86 cell surface marker. An overview of the viability and CD86 cell surface marker activity is summarized in Table 1 (attached). The results of the positive, negative and vehicle controls are summarized in Table 2. An overview of EC150 and CV70 values is given inTable3. The individual raw data are presented in Appendix 2 (attached).

 

Two independent experiments were performed. The cell viability before incubation with the test item was > 90% (99% and 97% in experiment 1 and 2, respectively). The cells were in these experiments incubated with Bis-Aminopropyl Diglycol Dimaleate in a concentration range of 1.0 – 200 µg/mL. The increase of CD86 cell surface marker expression was assessed by measuring the amount fluorescent cell staining of the CD86 cell surface marker compared to the solvent control. In addition, the viability was assessed with propidium iodide.

 

Experiment 1

· No precipitation was observed at the end of the incubation period in the 96-well plates.

· Bis-Aminopropyl Diglycol Dimaleate showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 µg/mL. 

· A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150is 2.8 µg/mL.

· The test item showed colour interference at 100 and 200 µg/mL.

· The positive control (TNBS) showed a S.I. ≥ 573% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).The negative control (LA)showed a S.I. ≤ 104% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

 

Experiment 2

· No precipitation was observed at the end of the incubation period in the 96-well plates.

· Bis-Aminopropyl Diglycol Dimaleate showed toxicity, the calculated CV70was 9.5 µg/mL.

· A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150is 182 µg/mL.

· The test item showed colour interference at 100 and 200 µg/mL.

The positive control (TNBS) showed a S.I. ≥ 305% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).The negative control (LA)showed a S.I. ≤ 112% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

 

Discussion

The test item showed only toxicity in experiment 2 (CV70 value of 182 µg/mL in experiment 2). A biologically relevant, induction of the CD86 activity (EC 150 values of 2.8 µg/mL and 9.5 µg/mL in experiment 1 and 2, respectively) was measured in both experiments.

Bis-Aminopropyl Diglycol Dimaleate is classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

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, Bis-Aminopropyl Diglycol Dimaleate is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.
Executive summary:

The objective of this study is to evaluate the ability of Bis-Aminopropyl Diglycol Dimaleate to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay.

 

The study procedures described in this report were based on the most recent OECD guideline (OECD 422E).

 

Bis-Aminopropyl Diglycol Dimaleate was a clear to transparent yellow liquid. A correction factor of 3.85 was used to correct for the water content (74%). The test item was dissolved in complete medium at 0.4 mg/mL. In the first experiment the stock was diluted to six test concentrations (1, 10, 20, 50, 100 and 200μg/mL). In the second experiment, a more narrow dose-response analysis was performed to investigate the increase in expression of experiment 1 in more detail. No precipitate was observed at any dose level tested. Two independent experiments were performed.

 

Both experiments passed the acceptance criteria:

· At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (99% in experiment 1 and 98% in experiment 2).

· The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in both experiments.

· At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5% in both experiments.

· No drift in CD86 expression was observed in the untreated controls and negative controls.

 

In both experiments the positive and negative control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

 

The test item showed only toxicity in experiment 2 (CV70value of 182µg/mL in experiment 2). A biologically relevant, induction of the CD86 activity (EC150values of 2.8 µg/mL and 9.5 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. Bis-Aminopropyl Diglycol Dimaleate is classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

 

In conclusion, Bis-Aminopropyl Diglycol Dimaleate is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14 August 2017 to 31 October 2017 and 7 November 2017 to 14 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
OECD Guideline TG 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method (adopted February, 2015).
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
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.
Positive control results:
The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
The EC1.5 of the positive control was between 5 and 125 µM (43 µM and 63 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.57-fold and 2.35-fold in experiment 1 and 2, respectively).
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
EC 1.5 [442D]
Remarks:
Study 20140925
Value:
96 µM
Remarks on result:
other: Imax = 3.17
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
EC 1.5 [442D]
Remarks:
Study 518597
Value:
757 µM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Imax = 1.76
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
EC 1.5 [442D]
Remarks:
Study 518597
Value:
1 427 µM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Imax = 1.51
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
EC 1.5 [442D]
Remarks:
Study 20140925
Value:
269 µM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Imax = 2.36

The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (4.8% and 5.9% in experiment 1 and 2, respectively).

Interpretation of results:
study cannot be used for classification
Remarks:
See RSS endpoint: Skin sensitisation, WoE, Charles River (2020); Weight of Evidence
Conclusions:
2018 Study
In conclusion, based on the current study and the two experiments performed in Study No. 518597, Bis-Aminopropyl Diglycol Dimaleate is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) in the KeratinoSensTM assay.

2017 Study
The test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (EC1.5 values of 1048 µM and 1975 µM in experiment 1 and 2, respectively) was measured at concentrations lower than 1000 µM in both experiments. The maximum luciferase activity induction (Imax) was 1.76-fold and 1.51-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSensTM assay since in both experiments no biological relevant luminescence induction at concentrations below 1000 µM was observed. In conclusion, Bis-Aminopropyl Diglycol Dimaleate is classified as negative (no biological relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Executive summary:

2018 Study

The objective of this study was to evaluate the ability of Bis-Aminopropyl Diglycol Dimaleate to activate the antioxidant / electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay.

 

In Test Facility Study No. 518597 two KeratinoSens experiments were performed. One assay run gave a positive result (EC1.5of 757 µM) and the second assay run gave a negative result (EC1.5of 1427 but no induction below 1000 µM). At first instance the conclusion of Test Facility Study No. 518597 was negative but due to a change in the correction factor used for the correction of the water content no conclusion about the classification was possible since the results of the two experiments in Test Facility Study No. 518597 were not concordant. A third assay run (experiment) was required for a classification in the KeratinoSensTM assay. This assay run is performed in the current study.

 

The study procedures described in this report were based on the most recent OECD guideline.

 

Batch 11_13_17_1 of Bis-Aminopropyl Diglycol Dimaleate was a clear to transparent yellow liquid. A correction factor of 3.85 was used to correct for the water content (74%). Bis-Aminopropyl Diglycol Dimaleate was dissolved in dimethyl sulfoxide at 76 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of0.38 – 776 µM (2-fold dilution series). 

No precipitate was observed at any dose level tested. One experiment was performed.

 

The experiment passed the acceptance criteria:

- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. 

- The EC1.5of the positive control was between 5 and 125 µM (96 µM). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.17-fold).

- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (6.6%).

 

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

 

Bis-Aminopropyl Diglycol Dimaleate showed no toxicity (no IC30and IC50value) in the current experiment. This result is similar to the results that were observed in the two experiments performed in Test Facility Study No.518597.

A biologically relevant, dose-related induction of the luciferase activity (EC1.5value 269 µM) with a maximum induction of 2.36-fold was measured in the current experiment. In Test Facility Study No. 518597 two KeratinoSens runs were performed. One assay run gave a positive result (EC1.5of 757 µM) and the second assay run gave a negative result (EC1.5of 1427 µM but no induction below 1000 µM).

 

Bis-Aminopropyl Diglycol Dimaleate is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed in 2 out of 3 experiments at test concentrations ≤ 1000 µMwith a cell viability >70%.

In conclusion, based on the current study and the two experiments performed in Test Facility Study No. 518597, Bis-Aminopropyl Diglycol Dimaleate is classified as positive (activation of the antioxidant / electrophile responsive element (ARE)-dependent pathway in keratinocytes) in the KeratinoSensTM assay.

 

2017 Study

The objective of this study was to evaluate the ability of Bis-Aminopropyl Diglycol Dimaleate to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay.

The study procedures described in this report were based on the most recent OECD guideline.

 

Batch 3 of the test item was a clear colourless to pale yellow liquid. A correction factor of 2.778 was used to correct for the water content (74% water). The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution steps) in experiment 1 and of 172 – 2000 µM (1.25-fold) in experiment 2. In the second experiment, a more narrow dose-response analysis was performed using a lower dilution factor of 1.25-fold to investigate the induction at 1000 µM in experiment 1 in more detail. Two independent experiments were performed.

 

Both experiments passed the acceptance criteria:

- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.

- The EC1.5 of the positive control was between 5 and 125 µM (43 µM and 63 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.57 -fold and 2.35-fold in experiment 1 and 2, respectively).

- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (4.8% and 5.9% in experiment 1 and 2, respectively).

 

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

 

The test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (EC 1.5 values of 1048 µM and 1975 µM in experiment 1 and 2, respectively) was measured at concentrations lower than 1000 µM in both experiments. The maximum luciferase activity induction (Imax) was 1.76-fold and 1.51-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSensTM assay since in both experiments no biological relevant luminescence induction at concentrations below 1000 µM was observed.

 

In conclusion, Bis-Aminopropyl Diglycol Dimaleate is classified as negative (no biological relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint:
skin sensitisation, other
Remarks:
Weight of Evidence - Skin Sensitization of bis-aminopropyl diglycol dimaleate (CAS No. 1629579-82-3)
Type of information:
other: Weight of Evidence - Skin Sensitization of bis-aminopropyl diglycol dimaleate (CAS No. 1629579-82-3)
Adequacy of study:
weight of evidence
Study period:
08 June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 - Weight of Evidence report
Qualifier:
no guideline required
Principles of method if other than guideline:
A Weight of Evidence approach has been used to address this endpoint, information based on the structure and the reactivity of the individual components of bis-amino diglycol dimaleate, the maleate groups are of concern for possible skin sensitizing properties were assesssed.
GLP compliance:
no
Type of study:
other: Weight of Evidence
Specific details on test material used for the study:
Test Item Name: Bis-Aminopropyl Diglycol Dimaleate
CAS Number: 1629579-82-3
Key result
Parameter:
other: Weight of Evidence
Remarks on result:
not determinable
Remarks:
More information on the sensitizing potency of the formulation(s) is needed to demonstrate non skin sensitizing properties for the formulation(s).

EXPERIMENTAL DATA

- Results of studies with bis-aminopropyl diglycol dimaleate

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. Bis-aminopropyl diglycol dimaleate is predicted to be not sensitizing to the skin.

A valid DPRA test was performed according to OECD 442C and in accordance with GLP principles. Bis-aminopropyl diglycol dimaleate was dissolved in acetonitrile at 72 mM and formed a clear solution by visual inspection. No co-elution of the test item with SPCC or SPCL was observed. In the cysteine reactivity assay the test item showed 92.3% SPCC depletion, and in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 46.2%. 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 and was considered to be positive in the DPRA. Due to an error in the initial correction factor used during the study, the prepared test item stock solution had a concentration of 72 nM instead of the desired 100 nM. However, since a positive result and “high reactivity” classification was already obtained at this lower test item concentration, this calculation error had no impact on the outcome of the study.

Two KeratinoSensTM assays were performed according to OECD 442D and in accordance with GLP principles. Bis-aminopropyl diglycol dimaleate was dissolved in DMSO. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay. The test item concentrations used in the first study were corrected based on a purity of 36% (factor 2.778). However, it appeared that the test item concentrations should have been corrected for a purity of 26% and as a consequence the actual concentrations tested in this first study were lower resulting in test concentrations up to and including 757 μM (2-fold dilution steps) in experiment 1 and up to and including 1427 μM (1.25-fold) in experiment 2. The test item showed no toxicity (no IC30 and IC50 value) in experiment 1 and 2. A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value 757 μM) with a maximum induction of 1.76-fold was measured in the first experiment. No biologically relevant induction of the luciferase activity was measured in experiment 2 (EC1.5: 1427 μM; Lmax 1.51). No conclusion about the classification was possible since the results of the two experiments were not concordant (experiment 1 positive, experiment 2 negative). A third assay experiment was needed for a final conclusion which was performed in a second study. In this second study bis-aminopropyl diglycol dimaleate was dissolved in dimethyl sulfoxide at 76 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.38 – 776 μM (2-fold dilution series). No precipitate was observed at any dose level tested. One experiment was performed. Bis-aminopropyl diglycol dimaleate showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value 269 μM) with a maximum induction of 2.36-fold was observed in the third experiment. In conclusion, bis-aminopropyl diglycol dimaleate is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed in 2 out of 3 experiments at test concentrations ≤ 1000 μM with a cell viability >70%.

A valid U-SENSTM assay was performed according to OECD 442E and in accordance with GLP principles. Bis-aminopropyl diglycol dimaleate was dissolved in complete medium at 0.4 mg/mL. In the first experiment the stock was diluted to six test concentrations (1, 10, 20, 50, 100 and 200 μg/mL). In the second experiment, a more narrow dose-response analysis was performed to investigate the increase in expression of experiment 1 in more detail. No precipitate was observed at any dose level tested. Two independent experiments were performed. Bis-aminopropyl diglycol dimaleate showed only toxicity in one experiment (CV70 value of 182 μg/mL in experiment 2). A biologically relevant, induction of the CD86 activity (EC150 values of 2.8 μg/mL and 9.5 μg/mL in experiment 1 and 2, respectively) was measured in both experiments. Bis-aminopropyl diglycol dimaleate is classified as positive in the U-SENSTM assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

Human repeat insult patch tests (HRIPT) were performed for skin irritation and skin sensitization evaluation of 3 formulations which contain Bis-aminopropyl diglycol dimaleate as active ingredient. Olaplex Bond Multiplier No.1 (20% bis-aminopropyl diglycol dimaleate) was tested under occlusive conditions in 67 subjects and Olaplex Bond Perfector No.2 (3% bis-aminopropyl diglycol dimaleate) and Olaplex Hair Perfector No.3 (3% bis-aminopropyl diglycol dimaleate) were tested in 50 subjects under semi-occlusive conditions (Bioscreen 2015a,b). Approximately 0.02-0.05 mL of the test material was dispensed on a 7.5 mm paper disk were then affixed directly to the skin of the intrascapular regions of the back. Subject were instructed to remove the patches approximately 48 hr after the first application and 24 hr thereafter for the remainder of the study. This procedure was repeated until a series of nine consecutive 24 hr exposures had been made three times a week for three consecutive weeks.

Following a 10-14 day rest period, a 24-hr challenge application was made to a previously untreated site in the same manner as the induction applications, and reactions were scored 48 and 96 hr after application. No adverse reactions of any kind were reported during the course of the studies for any of the three formulations. The researchers concluded that there were no identifiable signs or symptoms of primary irritation or sensitization for Olaplex Bond Multiplier No.1, Olaplex Bond Perfector No.2 and Olaplex Hair Perfector No.3.

- Results of studies with maleic acid

Maleic acid has a harmonized classification Skin Sens 1 according to Regulation (EC) No 1272/2008 with a specific concentration limit ≥ 0.1%. The REACh dossier disseminated file (https://echa.europa.eu/registration-dossier/-/registered-dossier/15769) describes that two skin sensitisation studies are available for maleic acid, a positive LLNA (2004, not publicly available) and a positive GPMT (2002, not publicly available, minimal number of animals included and borderline positive result).

Kreiling et al (2008) investigated the skin sensitizing potential of eight unsaturated and one saturated lipid (bio)chemical including maleic acid in both the local lymph node assay (LLNA) and the guinea pig maximization test (GPMT). It was hypothesized that chemicals with unsaturated carbon–carbon double bonds may result in a higher number of unspecific (false positive) results in the LLNA compared to the GPMT. The LLNA was performed according to OECD TG 429 (OECD 2002). Maleic acid was tested at 10%, 25% and 50% resulting in stimulation indices (SIs) of 6.7, 16.1 and 16.1, respectively. As the SI was ≥3 the result was considered positive. The GPMT was performed according to OECD TG 406 (OECD 1992). The test concentrations used were 0.5% for intradermal induction and 25% for topical induction and challenge. A grade one skin reaction was observed in one (out of 10) animals of the treatment group after the first challenge. A re-challenge was performed and again one animal showed a grade one skin reaction. The animal that had reacted after the first challenge failed to react after the re-challenge and this reaction was therefore not reproducible

and the animal was not considered sensitized. The GPMT was concluded to be negative.

Kreiling et al (2017) investigated whether the non-animal test methods, either on their own or integrated by a prediction model, have similar limitations with respect to the testing of chemicals with unsaturated carbon–carbon double bonds as the LLNA. Maleic acid tested positive (high reactivity) in the DPRA. In the KeratinoSensTM assay maleic acid tested negative in the first run, positive in the second run (EC1.5 could not be determined) and negative in the third run leading to an overall negative result. In the h-CLAT maleic acid  tested negative in the first run, positive in the second run (RFI = 210 for CD54 at 279 µg/mL) and negative in the third run leading to an overall negative call. Taken into account the results for the DPRA, KeratinoSensTM and the h-CLAT, maleic acid was predicted to be a non-sensitizer based on the 2-out-of-3 prediction model (Kreiling et al 2017). The LLNA is the in vivo method against which the in chemico/in vitro assays had generally been validated (Dumont et al., 2016). Based on the evaluation of the outcome of the 2-out-of-3 prediction model (Kreiling 2017) and comparison to GPMT and LLNA data which was performed for eight unsaturated and one saturated lipid (bio)chemicals (Kreiling 2008), it was concluded by Kreiling et al (2017) that the 2-out-of-3 prediction model was over-predictive of skin sensitizing effects of the eight tested unsaturated and one saturated lipid (bio)chemicals.

Frohwein et al (2016) tested the same set of substances which had been tested by Kreiling et al (2008) using a combined in vitro assay for sensitizing and irritative potential: the Loose-fit Coculture-based Sensitization Assay (LCSA; Schreiner et al (2007)). The estimated concentration of maleic acid to elicit half-maximal CD86 increase on dendritic cells in the LCSA was 1983 μmol/L and the strength of sensitizing potential in the LCSA was therefore classified as ‘weak’ (>100 μmol/L). The authors concluded that considering the widespread use of maleate salts in pharmaceutical formulations, the sensitizing potency for maleic acid, if any, should be very low (Frohwein et al 2016) and that the sensitizing potential of maleic acid is more accurately measured by the LCSA than by the LLNA.

DISCUSSION

A positive DPRA, a positive KeratinoSensTM assay and a positive U-SENSTM assay suggest that bis-aminopropyl diglycol dimaleate may have skin sensitizing properties. The Derek QSAR prediction was negative as the structures of bis-aminopropyl diglycol dimaleate do not match the constrains for alert 480: alpha,beta-unsaturated ketone or precursor. However, this alert is based on mechanistic studies that indicate activity through a mechanism of Michael addition of skin nucleophiles such as protein cysteine thiol groups which is the proposed working mechanism for bis-aminopropyl diglycol dimaleate. The negative Derek prediction is therefore considered less reliable. Based on the structure and the reactivity of the components present in bis-aminopropyl diglycol dimaleate, the maleate groups are expected to be responsible for the effects observed in the in chemico/in vitro tests performed. Comparison of the available skin sensitisation data for bis-aminopropyl diglycol dimaleate and maleic acid supports the hypothesis that the maleate groups in bis-aminopropyl diglycol dimaleate are likely responsible for the observed effects.

Similar to a DPRA performed with maleic acid (Kreiling et al 2017), bis-aminopropyl diglycol dimaleate has a high reactivity towards the cysteine-peptide and was classified in the high reactivity class when using the Cysteine 1:10 / Lysine 1:50 prediction model. This high reactivity towards cysteine which contains a sulfur atom is expected given the application of bis-aminopropyl diglycol dimaleate in cosmetics products to repair disulphide bonds in hair.

The KeratinoSensTM assay performed with bis-aminopropyl diglycol dimaleate was positive based on two positive runs and one negative run with EC1.5 values at 757 μM, 269 μM and 1427 μM. For maleic acid, two negative and one positive run led to an overall negative result in the KeratinoSensTM assay (Kreiling et al 2017). For both substances a concentration-dependent increase in cell viability was observed up to 150 to 175% of control level. In the U-SENSTM assay, bis-aminopropyl diglycol dimaleate increased the CD86 expression leading to a positive test result. An increase in CD86 was also described for maleic acid in a loose-fit coculture-based sensitization assay by Frohwein et al (2016), suggesting that exposure to both maleic acid and bis-aminopropyl diglycol dimaleate leads to dendritic cell activation.

Overall, the available data for maleic acid include two positive LLNA’s (EC3 <1% and <10%), one borderline positive and one negative GPMT, a negative 2-out-of-3 model prediction for skins sensitisation based on in chemico/in vitro tests and a ‘weak’ classification based on an alternative in vitro test. The in vitro data suggest that the LLNA possibly overestimates the skin sensitisation potency of maleic acid but there is no sufficient evidence to conclude that classification of maleic acid is not warranted.

The alternative experimental data available for bis-aminopropyl diglycol dimaleate consists of a negative DEREK, positive DPRA, positive KeratinoSens TM assay and positive U-SENS. There is no in vivo study (LLNA or GPMT) available. Because the alternative data on bis-aminopropyl diglycol dimaleate suggest the substance has sensitising properties and the available literature on maleic acid does not provide sufficient evidence that maleic acid is ‘overclassified’, it cannot be excluded that bis-aminopropyl diglycol dimaleate has skin sensitizing properties.

According to the United Nations' Globally Harmonised System of Classification and Labelling of Chemicals (GHS) mixtures can be classified on a weight of evidence evaluation of reliable and good quality evidence from human experience or appropriate studies in experimental animals. Three products containing bis-aminopropyl diglycol dimaleate in concentrations up to and including 20% showed no adverse reactions in HRIPT studies. However, these studies included a small number of subjects (50 to 67 while normally at least 100 is required (ECETOC 2002). Based on the concern for skin sensitizing potential identified in the in chemico/in vitro studies and the data available on maleic acid, the HRIPT

studies alone are not considered sufficient to conclude that the products containing bis-aminopropyl diglycol dimaleate up to and including 20% do not have to be classified.

Overall, considering all available information on skin sensitizing properties on both maleic acid and bis-aminopropyl diglycol dimaleate, there is no firm and reliable evidence that bis-aminopropyl diglycol dimaleate has no skin sensitizing properties. However, as it is the intention to demonstrate non skin sensitizing properties for the formulations and not for bis-aminopropyl diglycol dimaleate itself, there is the option to perform additional testing on the

product(s)/formulations, to demonstrate non skin sensitizing properties for the tested formulation. Actual data on skin sensitizing properties of the formulation generally overrules the results obtained with the calculation rules.

Interpretation of results:
other: More information on the sensitizing potency of the formulation(s) is needed to demonstrate non skin sensitizing properties for the formulation(s).
Conclusions:
Based on the in chemico/in vitro results and human data for bis-aminopropyl diglycol dimaleate and available data from public literature for maleic acid on skin sensitizing properties, it cannot be excluded that bis-aminopropyl diglycol dimaleate possess skin sensitization properties. The negative HRIPT data are not considered sufficient evidence that formulations containing bis-aminopropyl diglycol dimaleate up to and including 20% do not warrant classification and labelling of these products for skin sensitization. More information on the sensitizing potency of the formulation(s) is needed to demonstrate non skin sensitizing properties for the formulation(s).
Executive summary:

The objective of this evaluation was to reach an overall conclusion on the classification for skin sensitization of products containing bis-aminopropyl diglycol dimaleate based on all available relevant information, including in silico/in chemico/in vitro and human data. Available studies addressing the key events of sensitization as well as human data and relevant publications were evaluated and integrated in a weight of evidence approach for classification and labelling purposes.

A positive DPRA, a positive KeratinoSensTM assay and a positive U-SENSTM assay suggest that bis-aminopropyl diglycol dimaleate may have skin sensitizing properties, while the DEREK QSAR prediction was negative. Based on the chemical structure, the reactivity of the components and similarity in effects observed in chemico and in vitro between maleic acid and bis-aminopropyl diglycol dimaleate, the maleate groups are expected to be responsible for the effects observed in the in chemico/in vitro tests performed.

Maleic acid has a harmonized classification Skin Sens 1 according to Regulation (EC) No 1272/2008 with a specific concentration limit ≥ 0.1%. The available data for maleic acid includes two positive LLNA’s (EC3 <1% and <10%, respectively), one borderline positive and one negative GPMT, a negative 2-out-of-3 model prediction for skins sensitisation based on in chemico/in vitro tests and a ‘weak’ classification based on a non-guideline in vitro test.

The in vitro data are not in line with the results of the LLNA test and suggest that the LLNA possibly overestimates the skin sensitisation potency of maleic acid but there is not sufficient evidence to conclude that classification of maleic acid is not warranted. Three products containing bis-aminopropyl diglycol dimaleate in concentrations up to and including 20% showed no adverse reactions in a HRIPT study. However, these studies included a small number of subjects (50 to 67 while normally at least 100 is required). Based on the concern for skin sensitizing potential identified in the in chemico/in vitro studies and the data available on maleic acid, the HRIPT studies alone are not considered sufficient evidence to conclude that the products containing bis-aminopropyl diglycol dimaleate up to and including 20% do not have skin sensitizing properties and do not need to be classified.

In conclusion, based on the in chemico/in vitro results and human data for bis-aminopropyl diglycol dimaleate and available data from public literature for maleic acid on skin sensitizing properties, it cannot be excluded that bis-aminopropyl diglycol dimaleate possess skin sensitization properties. The negative HRIPT data are not considered sufficient evidence that formulations containing bis-aminopropyl diglycol dimaleate up to and including 20% do not warrant classification and labelling of these products for skin sensitization. More information on the sensitizing potency of the formulation(s) is needed to demonstrate non skin sensitizing properties for the formulation(s).

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)
Additional information:

Based on the in chemico/in vitro results and human data for bis-aminopropyl diglycol dimaleate and available data from public literature for maleic acid on skin sensitizing properties, it cannot be excluded that bis-aminopropyl diglycol dimaleate possess skin sensitization properties. The negative HRIPT data are not considered sufficient evidence that formulations containing bis-aminopropyl diglycol dimaleate up to and including 20% do not warrant classification and labelling of these products for skin sensitization. More information on the sensitizing potency of the formulation(s) is needed to demonstrate non skin sensitizing properties for the formulation(s).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification