trisodium 3-[{3-[bis(2-carboxylatoethyl)amino]propyl}(C12-18-(even numbered) and C18-(unsaturated) alkyl)amino]propanoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 30 October 2017. Experimental completion date: 07 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

yes

Remarks:

Please see any other information on materials and methods section

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Skin sensitisers have been reported to induce genes that are regulated by the antioxidant response element (ARE). The KeratinoSens™ test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laborat ory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSens™ test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The method cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories lA and lB as defined by the UN GHS, for authourities implementing these two optional subcategories, nor to predict potency for safety assessment decisions. However, depending on the regulatory framework a positive result may be used on its own to classify a chemical int o UN GHS category 1.

Test material

Test material information

Constituent 1

Reference substance name:

trisodium 3-[{3-[bis(2-carboxylatoethyl)amino]propyl}(C12-18-(even numbered) and C18-(unsaturated) alkyl)amino]propanoate

EC Number:

701-354-5

Molecular formula:

Not applicable UVCB

IUPAC Name:

trisodium 3-[{3-[bis(2-carboxylatoethyl)amino]propyl}(C12-18-(even numbered) and C18-(unsaturated) alkyl)amino]propanoate

Test material form:

liquid

Details on test material:

Product name: Sodium cocopropylenediamine propionate
CAS no (old) : 97659-50-2
CAS no (new): 2136366-30-6
Batch no.: 48724
Date of Production: 18.05.2017
Best before Date: 17.05.2020
Purity (certified): 29.7% w/w (UVCB) - Total solids (activity) % w/w 29.7 Lower limit: 29.0, Upper limit: 30.0

Main active ingredients
Dodecylpropylenediamine tripropionate: 64% w/w
Tetradecylpropylenediamine tripropionate: 16% w/w
(considering the composition of the other constituents it is considered justified that the two main constituents represent the whole test item)

Water solubility: soluble
Appearance: yellow, clear
State:liquid
Stability under test conditions: not specified

Viscosity at 20°C Cps 34, Upper limit:150
Color (20% aq solution) Hu 100, Upper limit: 250
pH (20% aq solution) pH 6.3, Lower limit: 6.0, Upper limit: 7.0
Recommended storage
Store container tightly closed in a dry, well-ventilated place

Specific details on test material used for the study:

Test Item Name: Sodium CPDP (sodium Cocopropylenediamine propionate)
Cas Number: 97659-50-2
Purity: ~30% (from CoA)
Expiry date: 14 Dec 2019
Physical state: Liquid
Storage conditions: Room temperature dark

In vitro test system

Details on the study design:

Method of administration of test item:
A single application of 12 concentrations (2000, 1000,  500,  250, 125, 62.5, 31.25, 15.63, 7.81,  3.91, 1.95, 0.98 µM) of test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing.

Method of administration of reference items:
A single application of 5 concentrations (8, 16, 32, 64, 128 µM) of the positive control (Cinnamic Aldehyde) was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%.
A single application of culture medium with 1% DMSO was applied as the negative control.

Exposure times of test items and reference items:
Cells were incubated with the test or reference item for 48 ± 2h before endpoints  measurements.

Number of repetitions:
Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence and 2 x 96-well plates for MTT. The validity of each repetition was assessed following acceptance criteria described.

Overview
Preliminary testing: Determination of the top concentration by solubility testing
Day 1: Seeding cells (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT).
Day 2: 24h after seeding the test and control items were applied and plates were incubated at 37°C, 5% CO2, 2: 95% relative humidity for 48 ± 2h.
Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by Mn testing (2 plates)

Data Analysis
XCellR8 Forms F0056 A and B: Data Analysis for KeratinoSens™ (version 03) were used to analyse data. These forms are Microsoft Excel workbooks containing formulae to process the raw data as described in SOP L0057. The spreadsheets have been validated in-house (July 2017).

The following parameters were calculated in the KeratinoSens™ test method:
• the maximal average fold induct ion of luciferase activity (lmax) value observed at any concentration of the test item and positive control;
• the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5-fold threshold (i.e. 50% enhanced luciferase activity);
• For each concentration showing > 1.5-fold luciferase activity induction, statistical significance is calculated (e.g. by a two-tailed Student's t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.s determining concentration.
• The percentage of viability as compared to the Negative control

Results and discussion

Positive control results:

Positive Control (PC) (Cinnamic aldehyde) induced ≥1.5-fold in at least one concentration.
The average induction of cinnamic aldehyde was 1.91

In vitro / in chemico

Results

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Solubility
The test concentrations of Sodium CPDP used in the KeratinoSens™ test were selected on the basis of solubility test carried out prior to the study:
Solubility results of Sodium CPDP indicated the item was soluble in Cell Culture Medium at 200mg/ml. Testing on this study were done with subsequent dilution in cell culture medium 1% DMSO giving a top
concentration of 2000µM.

Discussion
The human skin sensitisation potential of Sodium CPDP was assessed using validated in vitro method: the KeratinoSens™ test to determine keratinocyte activation. The method was adapted to animal product-free conditions by XCellR8 and reference chemicals described in the guideline and in the performance standards were used to confirm the reliability, accuracy, sensitivity and specificity values.
The adapted method showed full concordance with the Validated Reference Method (VRM) - the KeratinoSens™ standard protocol. We recently obtained clarification from the European Chemicals
Agency (ECHA) that data using the adapted method may be used in REACH submissions, provided that the Performance Standards data, demonstrating equivalence with the VRM, is included in the dossier.
In this study, Sodium CPDP was classified as non-sensitiser to human skin. The sensitisation potential of Sodium CPDP was quantified by calculating 2 parameters known as the EC1.% and the IMAX value. The meanings of these are as follows:
• The EC1.s value means the Effective Concentration (EC) of test item that caused an induction of luciferase activity of greater than 1.5-fold over untreated controls. If at least one concent ration induces luciferase activity to ~1.5, then the product is classified as a skin sensitiser. (Note: this classification also requires the cell viability measured by MTI to be greater than 70%). Sodium CPDP caused luciferase induction ~1.5 in repetition 2, at 15.625µM, however th ere was no dose-response induction and Sodium CPDP was classified as a non-sensitiser.
• The Imax value is the maximum-fold induction observed within the concentration range tested. Although the KeratinoSens™ test is not validated to predict potency, the Imax value can provide a useful tool for a very preliminary comparison of sensitisation potential between test items. As shown in Section 13.2, the maximum induction was observed at a test concentration of 125µM, which showed an lmax value of 1.275 in repetition 1; 1.807 at 15.625µM in repetition 2; 1.375 at 7.813µM in repetition 3. For reference, during test validation, sensitising proficiency chemicals produced lmax values of up to 36-fold over untreated controls.

Any other information on results incl. tables

Determination criteria for the skin sensitisation potential of Sodium CPDP

	REP 1	REP 2	REP 3
Does at least one concentration of Test Item induce luciferase activity ≥ 1.5-fold:	No	Yes	No
Does the first concentration inducing luciferase activity above 1.5,have a viabilityabove 70%:	N/A	Yes(118.81%)	N/A
Does EC1.5 value occur at a concentration <1000µM(or <200µg/ml)	N/A	Yes(10.32µM)	N/A
Doesthe test item induce the luciferaseina dose- dependent manner	N/A	No	N/A
Classification	Non-Sensitiser	Non-Sensitiser	Non-Sensitiser

Assay Acceptance Criteria (Mean of the 3 repetitions)

Criteria	Result	Pass or Fail
1 -Positive Control (PC) (Cinnamic aldehyde) induction ≥1.5-fold in at least one concentration	Yes	Pass
2-Average induction of PC at 32µ M is (1.6-3.0)	Yes(1.91)	Pass
3-EC1.5 value is [6-39µM]	Yes (10.25)	Pass
4-CV% of blank values < 20%	9.72	Pass

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study, Sodium CPDP was classified as non-sensitiser to human skin.

Executive summary:

The human skin sensitisation potential of Sodium CPDP was assessed using the validated in vitro method, the KeratinoSens ™ assay, adapted to fully animal-free by XCellR8, and validated in-house to determine keratinocyte activation. After 48h exposure of cells with 12 concentrations of Sodium CPDP, Luciferase measurements and MTT viability testing were performed.

Sodium CPDP was classified as non-sensitiser.