Registration Dossier

Administrative data

Description of key information

Three in-vitro studies, DPRA, KerationSens and h-CLAT have been performed on the current test substance which is imported as a ca. 30% solution in water.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
January - February 1993
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This study is considered not to be reliable as we have no details of the composition of the test substance. Testing was done on a development lab prepared sample, with high residuals and by-products. Significant process differences have taken place since this study and the product is now toll manufactured outside the EU and imported. Testing for REACH is on the current commercial product.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The default animal study for skin sensitisation, in the ECHA Guidelines, is the Local Lymph Node Assay (LLNA)OECD429, however it is accepted in the OECD guideline for the LLNA that it has some limitations, as follows: “Despite the advantages of the LLNA over TG 406, it should be recognised that there are certain limitations that may necessitate the use of TG 406 (13) (e.g. false negative findings in the LLNA with certain metals, false positive findings with certain skin irritants [such as some surfactant type chemicals] (19) (20), or solubility of the test substance)”. The test substance is a surfactant; therefore it may produce a false positive response in the LLNA test. The Klimisch 3 guinea pig study was done in 1993 when the LLNA was not fully accepted.
Specific details on test material used for the study:
These are not available, sample not representative of current commercial product.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Olac Ltd., Bicester, UK
- Age at study initiation: no info
- Weight at study initiation: no info
- Housing: in stainless steel cages with grid floors and tops, up to 5 per sex
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: no info


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-23
- Humidity (%): 40-70
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 January To: 26 February 1993
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Intradermal induction: 3% v/v in purified water and 5% v/v in FCA
Epicutaneous induction: 100% (undiluted)
Epicutaneous challenge: 50% and 10% v/v
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Intradermal induction: 3% v/v in purified water and 5% v/v in FCA
Epicutaneous induction: 100% (undiluted)
Epicutaneous challenge: 50% and 10% v/v
No. of animals per dose:
10 males + 10 females (test group)
5 males + 5 females (control group)
Details on study design:
RANGE FINDING TESTS:
Intradermal induction (2 males + 2 females): 1, 3, 5, 10, 30, and 50% v/v; 3% v/v (in water) and 5% v/v (in FCA) were selected
Topical induction (1 male + 1 female): 10, 30, 50% and undiluted; undiluted was selected
Topical challenge (2 males + 1 female): 10, 30, 50% and undiluted; 50% and 10% v/v (in water) were selected

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and topical)
- Exposure period: day 1 and day 8, respectively
- Test groups: yes
- Control group: no (vehicle)
- Site: scapular region
- Frequency of applications: see above
- Duration: single injection and 48 h occlusive application
- Concentrations: 3% (in water) and 5% v/v (in FCA), and undiluted, respectively

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 22
- Exposure period: 24 h (occlusive)
- Test groups: yes
- Control group: yes
- Site: flanks
- Concentrations: 50% and 10% v/v
- Evaluation (hr after challenge): 24 and 48 h after dressing removal

Scoring system:
0 no response
± barely perceptible erythema
1 slight erythema
2 moderate erythema
3 severe erythema



Challenge controls:
yes
Positive control substance(s):
no
Positive control results:
Not used
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50% v/v
No. with + reactions:
9
Total no. in group:
20
Clinical observations:
see below; control group 0/10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50% v/v . No with. + reactions: 9.0. Total no. in groups: 20.0. Clinical observations: see below; control group 0/10.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% v/v
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
see below; control group 0/10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50% v/v . No with. + reactions: 4.0. Total no. in groups: 20.0. Clinical observations: see below; control group 0/10.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10% v/v
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
see below
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10% v/v . No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: see below.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10% v/v
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
see below
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10% v/v . No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: see below.

Summary of dermal lesions (following sensitisation and topical challenge)

Animal no.

Days after challenge application

Score 100%
(undiluted)

Score 30%

1

2

50%

10%

50%

10%

211 M

0

0

0

0

-

-

212 M

1

0

± f

9

+

-

213 M

1

±

1 f

±

+

214 M

1

0

1

±

+

-

215 M

±

0

± f

0

-

-

216 M

±

0

±

0

-

-

217 M

±

0

0

0

-

-

218 M

1

0

±

0

+

-

219 M

1

0

±

0

+

-

220 M

0

0

0

0

-

-

221 F

1

0

1

0

+

-

222 F

±

±

±

±

-

-

223 F

±

±

±

±

-

-

224 F

1

0

±

0

+

-

225 F

±

0

±

0

-

-

226 F

±

0

0

0

-

-

227 F

1

0

1

0

+

-

228 F

1

0

±

0

+

-

229 F

±

0

0

0

-

-

230 F

±

0

0

0

-

-

0 no response; ± barely perceptible erythema; 1 slight erythema; f exfoliation;

+ = positive response; - = negative response

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the conditions of the present test, Ampholak YCE is a skin sensitiser however the test substance is considered not to be typical fo the current commencial product. The result is therefore not valid for the current Ampholak YCE.
Executive summary:

The potential of Ampholak YCE to cause delayed contact hypersensitivity in guinea-pigs was assessed by the Magnusson-Kligman Maximisation Test. The closely-cl ipped dorsa of ten male and ten female Dunkin-Hartley guinea-pigs were subject to intradermal injections of Freunds Complete Adjuvant, 3% v/v Ampholak YCE in purified water and 5% v/v Ampholak YCE in the adjuvant on Day 1. Seven days later the same area of skin was treated by topical application of Ampholak YCE as supplied and the test site was covered by an occlusive dressing for 48 hours. The same induction procedures were carried out on a contemporaneous control group of five male and five female animals, except that the test material was replaced by vehicle in all doses. On Day 22, all animals were challenged by occluded application of purified water to the left flank and Ampholak YCE at 50 and 10% v/v in purified water to two sites on the right flank. The occlusive dressings were removed on the following day and the condition of the test sites was assessed approximately 24 and 48 hours later. Intradermal injection of 3% v/v Ampholak YCE in purified water gave rise to slight or moderate erythema and discolouration. A similar administration of 5% v/v Ampholak YCE in the adjuvant caused moderate erythema, pallor, eschar formation and discolouration. Occluded topical application of Ampholak YCE as supplied gave rise to slight to moderate erythema, eschar formation, oedema and exfoliation. Challenge application of Ampholak YCE (50% v/v) caused a significant response (slight erythema or a more marked reaction) in 9/20 test animals but in 0/10 controls. No significant response was observed in in the animals following challenge application of 10% v/v Ampholak YCE in purified water or purified water alone. It was concluded that, under the conditions of this study and the criteria of OECD-GHS, repeated administration of Ampholak YCE caused delayed contact hypersensitivity in guinea-pigs.

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
Qualifier:
according to
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. certificate)
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.
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
Details on 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

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
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.

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
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.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 31 October 2017 Experimental completion date: 03 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
The reactivity and sensitization potential of Sodium cocopropylenediamine proprionate.
Specific details on test material used for the study:
Sponsor’s identification: Sodium cocopropylenediamine propionate
CAS No.: 97659-50-2
Batch No.: 48724
Purity: A 30% solution in water
Molecular weight: 477 g/mol
Appearance: Liquid, yellowish to brown
Retest date: 17 May 2020
Storage conditions: Ambient temperature (15-25°C)
Details on study design:
Peptide and Positive Control
Synthetic peptide containing Cysteine
Alternative name: Ac-RFAACAA-OH
Batch number: 1658140
Stated purity: 98% (by HPLC)
Molecular Weight: 751.7 g/mol
Supplier: AnaSpec
Storage conditions: Frozen (-10°C to -30°C)

Synthetic peptide containing Lysine
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Stated purity: 94% (by HPLC)
Molecular Weight: 776 g/moL
Supplier: AnaSpec
Storage conditions: Frozen (-10°C to -30°C)

Cinnamic Aldehyde (Positive control)
Batch number: MKCB9907
Stated purity: 99.1%
Molecular Weight: 132.16 g/moL
Supplier: SAFC
Storage conditions: Room temperature (15-25°C)
Expiry/retest date: November 2021

Apparatus
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Balances fitted with printers: Capability of weighing to 5 decimal places
General laboratory apparatus and glassware.

Analytical Procedure
Reagents
Acetonitrile (ACN): HPLC gradient grade
Trifluoroacetic acid (TFA): 99% Pure
Water: Deionised reverse osmosis
Ammonium Acetate: Analytical reagent
Sodium Phosphate, monobasic: Analytical reagent
Sodium Phosphate, dibasic: Analytical reagent
Ammonium Hydroxide: Analytical reagent
100 mM Phosphate buffer, pH 7.5: In house preparation
100 mM Ammonium Acetate buffer, pH 10.2: In house preparation
HPLC Mobile Phase A: 0.1% v/v TFA in Water, in house preparation
HPLC Mobile Phase B: 0.085% v/v TFA in ACN, in house preparation

Assessment of Test Item Solubility
The solubility of Sodium cocopropylenediamine proprionate in water was assessed at a concentration of 100 mM.

Preparation of Peptide Stock Solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.

Preparation of Stability Controls and Precision Controls
Stability controls (Reference Control B and Reference Control C) and precision controls of both peptides were prepared at a concentration of 0.5 mM. Reference Control B samples and the precision control sample contained acetonitrile. The reference control C was prepared with water replacing acetonitrile.

Preparation of Positive Control Solution and Test Item Stock Solution
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution in water of Sodium cocopropylenediamine proprionate was prepared.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Aqueous solutions of Sodium cocopropylenediamine proprionate and solutions of the positive control (in acetonitrile) were diluted with the Cysteine peptide to prepare solutions containing 0.5 mM Cysteine and 5 mM of either Sodium cocopropylenediamine proprionate or the positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co elution Controls
Aqueous solutions of Sodium cocopropylenediamine proprionate and solutions of the positive control were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either Sodium cocopropylenediamine proprionate or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the Sodium cocopropylenediamine proprionate and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of Sodium cocopropylenediamine proprionate and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

Instrumentation Parameters
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Zorbax SB C18, 3.5µm, 100 x 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30°C
Sample temperature: 25°C
Mobile Phase (MP) A: 0.1% TFA in Water
Mobile Phase (MP) B: 0.085% TFA in ACN
Gradient: Time (minutes) MP A (%) MP B (%)
0 90 10
20 75 25
21 10 90
23 10 90
23.5 90 10
30 90 10
Flow rate: 0.35 mL/minute
Stroke volume: 25 µL
Detector wavelength: UV, 220 nm
Injection volume: 2 µL (slow draw rate)
Run time: 30 minutes
Approximate retention time (Cysteine): 11 minutes
Approximate retention time (Lysine): 7 minutes

Calculations
The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:
% Peptide depletion = 100 - (Peptide peak area in replicate depletion samples (x 100)/Mean Peptide peak area of reference control samples B or C)
Positive control results:
Not applicable.
Parameter:
other: Overall mean depletion (%)
Run / experiment:
Mean
Value:
2.79
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation

Solubility Assessment

The solubility of Sodium cocopropylenediamine proprionate in water at a nominal concentration of 100 mM was confirmed.

Reactivity Assessment

All analytical acceptance criteria for each peptide run were met:

 

 

Peptide

Standard Linearity

Positive control depletion (%)

Reference controls

Test item

Acceptance criteria

Cysteine

r2>0.99

60.8-100
(SD <14.9%)

0.45-0.55 mM (CV <15%)

SD <14.9%

Lysine

r2>0.99

40.2-69.0
(SD <11.6%)

0.45-0.55 mM (CV <15%)

SD <11.6%

Achieved results

Cysteine

r2>0.999

70.1
(SD, 0.23%, n=3)

B: 0.500 mM (CV 1.06%, n=6)
C: 0.502 mM (CV 0.09%, n=3)

SD 0.90% (n=3)

Lysine

r2>0.999

55.1
(SD, 0.85%, n=3)

B: 0.503 mM (CV 0.28%, n=6)
C: 0.495 mM (CV 0.46%, n=3)

SD 0.42% (n=3)

CV         Coefficient of Variation

SD         Standard deviation

The depletion of peptide in the presence of Sodium cocopropylenediamine proprionate was:

Peptide

Mean peak area of reference control(µV.sec)

Mean peak area of peptide with test item(µV.sec)

Mean peptide depletion by Sodium cocopropylenediamine proprionate (%)

Cysteine

Control B: 861510 (n=6)

Control C: 863990 (n=3)

821300 (n=3)

4.94

Lysine

Control B: 760580 (n=6)

Control C: 749620 (n=3)

744780 (n=3)

0.646

Applying the following depletion model (below), reactivity is classed as “no to minimal” and the DPRA prediction is negative, hence Sodium cocopropylenediamine proprionateis predicted as a non-skin sensitizer. 

Mean of Cysteine and Lysine% depletion

Reactivity Class

DPRA Prediction

0%≤ mean% depletion ≤6.38%

No or minimal reactivity

Negative

6.38%< mean% depletion ≤22.62%

Low reactivity

Positive

22.62%< mean% depletion ≤42.47%

Moderate reactivity

42.47%< mean% depletion ≤100%

High reactivity

 

There was no co-elution peaks in either of the Lysine or Cysteine assays.   

Overall Achieved Depletion Values

Test item

Cysteine peptide depletion (%)

Lysine peptide depletion (%)

Overall mean depletion (%)

Reactivity class

DPRA prediction

Sodium cocopropylenediamine propionate 

4.94

0.646

2.79

No to minimal

Negative

Individual Achieved Depletion Values

Cysteine Peptide Depletion

Sample

Peak area (µV.sec)

Peptide concentration1
(µg/mL)

Peptide Depletion (%)

Mean Depletion (%)

SD
 (%)

Positive control

259091

113.504

69.92

70.1

0.23

257646

112.874

70.12

255151

111.794

70.42

Sodium cocopropylenediamine propionate 

827479

361.38

4.233

4.94

0.90

823887

359.81

4.643

812530

354.86

5.963

SD      Standard Deviation

1         Samples prepared at a concentration of 376 µg/mL (0.5 mM)

2         Calculated against a mean Reference Control B area of 861510 µV.sec(n=6)

3         Calculated against a mean Reference Control C area of 863990 µV.sec(n=3)

4         Individual and mean concentrations of the positive control are outside of the range established during the validation study. There is however considered to be no impact on the data reporting as both each individual and the overall mean depletion values are within the stated acceptance criteria of 60.8%-100% depletion.

Lysine Peptide Depletion

Sample

Peak area (µV.sec)

Peptide concentration1(µg/mL)

Peptide Depletion (%)

Mean Depletion (%)

SD
 (%)

Positive control

334244

169.92

56.12

55.1

0.85

345252

175.614

54.62

345547

175.764

54.62

Sodium cocopropylenediamine propionate 

747501

383.41

0.2833

0.646

0.42

741370

380.25

1.103

745475

382.37

0.5533

SD      Standard Deviation

1         Samples prepared at a concentration of 376 µg/mL (0.5 mM)

2         Calculated against a mean Reference Control B area of 760580 µV.sec(n=6)

3         Calculated against a mean Reference Control C area of 749620 µV.sec(n=3)

4         Individual concentrations of the positive control are outside of the range established during the validation study. There is however considered to be no impact on the data reporting as both each individual and the overall mean depletion values are within the stated acceptance criteria of 40.2% to 69.0% depletion.

Historic Control Data

Historic Data for Reference Controls

 

Cysteine peptide concentration (µg/mL)1

Lysine peptide concentration (µg/mL)2

Maximum

386

400

Minimum

364

382

Mean

378

393

Number

36

36

CV (%)

1.27

1.24

1             Samples prepared at a concentration of 376 µg/mL (0.5 mM)

2             Samples prepared at a concentration of 388 µg/mL (0.5 mM)

CV     Coefficient of Variation

Historic Data for Positive Controls

 

Cysteine peptide concentration (µg/mL)1

Lysine peptide concentration (µg/mL)2

Maximum

107

174

Minimum

101

153

Mean

1043

1624

Number

18

18

CV (%)

1.56

3.87

1          Samples prepared at a concentration of 376 µg/mL (0.5 mM)

2          Samples prepared at a concentration of 388 µg/mL (0.5 mM)

3      Overall mean depletion 72.6% (n=18)

4      Overall mean depletion 58.8% (n=18)

CV    Coefficient of Variation

Interpretation of results:
GHS criteria not met
Conclusions:
Solutions of Sodium cocopropylenediamine proprionate were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall mean depletion result of 2.79% places Sodium cocopropylenediamine proprionate in the reactivity class of “no to minimal activity” and hence it is predicted by DPRA to be a potential non-skin sensitizer.
Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of Sodium cocopropylenediamine proprionate. 

Solutions of Sodium cocopropylenediamine proprionate were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall result of 2.79% depletion places Sodium cocopropylenediamine proprionate in the reactivity class of “no to minimal” and therefore it is predicted by DPRA to be a potential non-skin sensitizer. 

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 18 October 2017 Experimental completion date: 07 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guidelines for the Testing of Chemicals: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), July 2016.
Deviations:
yes
Remarks:
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.
GLP compliance:
yes (incl. certificate)
Type of study:
other: Human Cell Line Activation Test (h-CLAT)
Justification for non-LLNA method:
An alternative method has been developed for the evaluation of the skin sensitisation potential by measuring phenotypic changes, such as CD86 and CD54 expression on dendritic cells. The human leukemia cell line THP-1 is used as surrogate for human myeloid dendritic cells, since these cells show also enhanced CD86 and/or CD54 expression when treated with sensitisers.
The purpose of the Human Cell Line Activation Test (h-CLAT) is to assess the skin sensitising potential of Sodium cocopropylenediamine propionate, CAS 97659-50-2 in an appropriate solvent (DMSO, saline or culture medium) when administered to THP-1 cells for 24 hours. The test item concentrations for the main experiment (h-CLAT) of Sodium cocopropylenediamine propionate, CAS 97659-50-2 are determined by XTT tests.
This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.
Specific details on test material used for the study:
Identification: Sodium cocopropylenediamine propionate, CAS 97659-50-2
Batch: 48724
CAS No.: 97659-50-2
Purity: 29.7% (w/w), the substance is a UVCB, purity is equal to total solids.*
Partition coefficient (n-octanol/water): log Pow: not indicated by the Sponsor
Water solubility: Soluble (not quantified)
Appearance: Liquid, yellowish to brown
Expiry Date: 17 May 2020
Storage Conditions: At room temperatue
Stability in Solvent: Stable in water (not quantified)
Purpose of Use: Industrial chemical
Details on study design:
Medium Control and Solvent Control for the Test Item
Name: Culture medium

Positive Control (h-CLAT)
Name: DNCB (2,4-dinitrochlorobenzene, CAS No.: 97-00-7) final concentration: 2 and 3 µg/mL, Purity ≥ 99%)
Solvent: DMSO

Solvent Control for the Positive Control (h-CLAT)
Name: DMSO (Dimethyl sulfoxide, CAS No. 67-68-5) in culture medium, final concentration 0.2%, Purity ≥ 99%

TEST ITEM PREPARATION
On the day of the experiment (immediately prior to start) Sodium cocopropylenediamine propionate, CAS 97659-50-2 was solved in culture medium.
The maximum concentration of test item was 5000 µg/mL culture medium, as tested by a solubility test.
For the XTT test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions from 5000 µg/mL culture medium. Due to strong cytotoxicity observed in the first XTT test, the test was repeated with adjusted test item concentrations. For this, dilutions were prepared by 1:2 serial dilutions from 50 µg/mL culture medium.

TEST SYSTEM AND SUPPORTING INFORMATION
Reasons for the Choice of THP-1 Cells
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.

THP-1 Cell Cultures
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH (aliquots of cells in freezing medium at 1x10^6 to 2x10^6 cells/mL) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1x10^6 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30).
The passage numbers of the used THP-1 cells were 17 and 13 in the valid XTT assays and 22 and 19 in the h-CLAT for runs 1 and 2, respectively.

Culture Medium
RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2 mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2 – 8 °C and used within one month. The culture medium has to be warmed to room temperature just before use.

Preparation and Seeding of THP-1 Cells
On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 µL with a cell density of 0.9 - 1x10^6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate.
For the main experiment (h-CLAT) 0.9 - 1x10^6 cells/well in a volume of 500 µL were seeded in a 24-well plate before the treatment. 

Experimental Design and Procedures of XTT
Dose Finding Assay (XTT Test)
The test item concentrations investigated in the main experiment (h-CLAT) were determined with two valid XTT tests.
The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl) - (3,4 - tetrazolium) – bis - (4 – methoxy – 6 - nitro) - benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. This method was first described 1988 by SCUDIERO et al. and improved in subsequent years by several other investigators.
Two valid independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT).

XTT Labelling Mixture
The XTT labelling mixture consists of two components, a XTT buffer solution and the substrate solution. Both components were mixed right before application at a ratio of 1:100.

Treatment
After the cell seeding, 100 µL of the test item dilutions, the medium and solvent controls, respectively, were added to the cells. All dose groups were tested in 7 replicates for each XTT test. At the end of the incubation period of 24 ± 0.5 hours, the cell cultures were microscopically evaluated for morphological alterations.

XTT Labelling and Measurement
At the end of the incubation period, 50 µL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Evaluation of the XTT results
A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. The relative absorbance (= viability in [%]) as compared to the solvent control is calculated using this formula:

Relative absorbance [%] = 100 x ((mean absorbance_test item - absorbance_chemical blank) / (mean absorbance_solvent control - absorbance_chemical blank))

To calculate the concentration of toxicant needed to reduce the relative absorbance to 75% of the solvent control (CV75) the following formula is used:

CV75 = Conc. >75 - ((Conc. >75 - Conc <75) * (% >75 - 75)) / (% >75 - %<75))

a) Conc. >75 = max. measured concentration with the % of solvent control >75%
b) Conc. <75 = min. measured concentration with the % of solvent control <75%
c) % >75 = relative absorbance at a) in %
d) % <75 = relative absorbance at b) in %

Calculation of the h-CLAT Test Item Concentrations
Two valid independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable CV75. The mean of at least two CV75 values was used to determine the dose-range for the main experiment (h-CLAT).
Eight final concentrations (µg/mL) were used for the test item in the main experiment (h-CLAT). The highest concentration used was 1.2 × CV75 and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.

Acceptability of the Assay
The XTT test is considered to be acceptable if it meets the following criteria:
• mean absorbance of the medium control is ≥ 0.5
• mean viability of the solvent control is ≥ 90% in comparison to the medium control

Experimental Design and Procedures of h-CLAT
The test item was tested in two independent runs.

Treatment of the Cells
For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment.
Each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

Staining of the Cells
The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 x g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 µL of blocking solution at 2 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).

Sample Preparation for Measurement
After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.

Flow Cytometry Acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

Preparation of the acquisition
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such that the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH).
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.

Acquisition
Dead cells were determined by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).
Positive control results:
The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.
Parameter:
other: RFI [%] - CD86
Run / experiment:
Mean
Value:
< 150
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
The result is considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP)
Parameter:
other: RFI [%] - CD54
Run / experiment:
Mean
Value:
< 200
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: the result is considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP)

Results of the Dose Finding Assay (XTT Test)

Results of the first XTT test for Test Item Sodium cocopropylenediamine propionate, CAS 97659-50-2

 

 

Microscopic Evaluation

Photometric Evaluation


Test Group

Concen-tration
[µg/mL]

Cytotoxicity

Mean Ab-sorbance*§

Standard-Deviation

Chem. Blank§

Mean Ab-sorbance – Chemical Blank

Absorbance in % of Solvent Control**

Medium Control

-

no

0.752

0.030

0.268

0.484

97.86

Solvent Control

-

no

0.751

0.026

0.256

0.495

100.00

Test Item

39.06

yes

0.238

0.002

0.238

0.000

-0.05

78.13

yes

0.240

0.008

0.245

-0.004

-0.90

156.25

yes

0.251

0.007

0.263

-0.012

-2.45

312.50

yes

0.272

0.018

0.290

-0.019

-3.75

625.00

yes

0.292

0.007

0.303

-0.011

-2.19

1250.00

yes

0.287

0.043

0.295

-0.008

-1.63

2500.00

yes

0.301

0.047

0.228

0.073

14.79

5000.00

yes

0.323

0.041

0.271

0.052

10.52

Shaded test groups: cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

§           Given absorbances are rounded values.

*           mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 102.2%.

Due to strong cytotoxicity in the XTT test, a CV75 value could not be calculated. The first XTT test was repeated with adjusted concentrations.

 Results of the repetition of the first XTT test for Test Item Sodium cocopropylenediamine propionate, CAS 97659-50-2

 

 

Microscopic Evaluation

Photometric Evaluation


Test Group

Concen-tration
[µg/mL]

Cytotoxicity

Mean Ab-sorbance*§

Standard-Deviation

Chem. Blank§

Mean Ab-sorbance – Chemical Blank

Absorbance in % of Solvent Control**

Medium Control

-

no

0.828

0.033

0.226

0.602

104.77

Solvent Control

-

no

0.808

0.060

0.234

0.574

100.00

Test Item

0.39

no

0.809

0.055

0.229

0.580

100.92

0.78

no

0.794

0.050

0.227

0.567

98.79

1.56

no

0.767

0.057

0.225

0.543

94.47

3.13

no

0.699

0.062

0.225

0.474

82.55

6.25

no

0.490

0.047

0.229

0.262

45.54

12.50

no

0.283

0.019

0.224

0.059

10.34

25.00

yes

0.263

0.025

0.211

0.052

9.04

50.00

yes

0.248

0.024

0.210

0.038

6.67

Shaded test groups: cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

§           Given absorbances are rounded values.

*           mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 95.5%.

The CV75 value of the first XTT test: 3.8 µg/mL


Results of the second XTT test for Test Item Sodium cocopropylenediamine propionate, CAS 97659-50-2

 

 

Microscopic Evaluation

Photometric Evaluation


Test Group

Concen-tration
[µg/mL]

Cytotoxicity

Mean Ab-sorbance*§

Standard-Deviation

Chem. Blank§

Mean Ab-sorbance – Chemical Blank

Absorbance in % of Solvent Control**

Medium Control

-

no

0.632

0.014

0.238

0.393

99.27

Solvent Control

-

no

0.639

0.014

0.243

0.396

100.00

Test Item

0.39

no

0.628

0.024

0.247

0.382

96.36

0.78

no

0.633

0.032

0.244

0.389

98.17

1.56

no

0.606

0.029

0.236

0.370

93.32

3.13

no

0.586

0.010

0.242

0.345

86.93

6.25

no

0.474

0.031

0.238

0.236

59.49

12.50

yes

0.286

0.016

0.240

0.046

11.59

25.00

yes

0.232

0.008

0.230

0.003

0.71

50.00

yes

0.226

0.010

0.220

0.006

1.54

Shaded test groups: cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

§            Given absorbances are rounded values.

*           mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 100.7%.

The CV75 value of the second XTT test: 4.5 µg/mL

The mean CV75 value of both XTT tests: 4.2 µg/mL


Results of the h-CLAT Test

Results of the first h-CLAT run for the Test Item Sodium cocopropylenediamine propionate, CAS 97659-50-2

 

Concentration (µg/mL)

RFI (%)
CD 54 Antibody

RFI (%)
CD 86 Antibody

Cell Viability (%)

 

Medium Control

-

100.0

100.0

100.0

DMSO Control

-

100.0

100.0

100.0

Positive Control (DNCB)

2.0

250.0*

332.9*

69.1

3.0

445.6*

519.4*

64.5

Test Item

1.4

101.8

84.2

92.6

1.7

108.2

87.3

91.5

2.0

112.7

96.9

91.6

2.4

100.0

78.0

95.7

2.9

101.8

78.6

94.4

3.5

96.4

80.7

95.7

4.2

105.5

88.5

91.5

5.0

145.5

107.8

80.1

*   RFI value of CD86 or CD54 fulfilled the positive criteria (CD86150% and CD54200%).

 

Results of the second h-CLAT run for the Test Item Sodium cocopropylenediamine propionate, CAS 97659-50-2

 

Concentration (µg/mL)

RFI (%)
CD 54 Antibody

RFI (%)
CD 86 Antibody

Cell Viability (%)

 

Medium Control

-

100.0

100.0

100.0

DMSO Control

-

100.0

100.0

100.0

Positive Control (DNCB)

2.0

267.9*

264.2*

84.3

3.0

367.2*

648.6*

67.2

Test Item

1.4

100.0

83.6

100.5

1.7

112.4

93.9

103.3

2.0

103.3

86.1

100.1

2.4

104.1

83.2

95.6

2.9

119.0

85.7

91.2

3.5

119.0

88.9

94.1

4.2

158.7

124.6

88.3

5.0

187.6

145.1

75.7

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86150% and CD54200%).

Results of the h-CLAT Test

Results of the first h-CLAT run for the Test Item Sodium cocopropylenediamine propionate, CAS 97659-50-2

 

Concentration (µg/mL)

Antibody / ISO

MFIGeoMean(FITC)

MFI-ISO

RFI (%)

Cyto(Geo)GeoMean(7-AAD)

Mean Cyto

Viability (%)

Medium control

-

ISO

2.14

 

 

3.82

3.6

100.0

CD54

3.24

1.10

100.0

3.80

CD86

5.36

3.22

100.0

3.10

DMSO control

-

ISO

2.09

 

 

4.06

3.7

100.0

CD54

3.23

1.14

100.0

3.71

CD86

4.98

2.89

100.0

3.26

Positive control (DNCB)

2

ISO

2.71

 

 

6.35

5.3

69.1

CD54

5.56

2.85

250.0

5.27

CD86

12.33

9.62

332.9

4.34

3

ISO

2.80

 

 

7.34

5.7

64.5

CD54

7.88

5.08

445.6

5.45

CD86

17.81

15.01

519.4

4.30

Test Item

1.4

ISO

2.32

 

 

4.29

3.9

92.6

CD54

3.44

1.12

101.8

3.89

CD86

5.03

2.71

84.2

3.40

1.7

ISO

2.34

 

 

4.35

3.9

91.5

CD54

3.53

1.19

108.2

3.93

CD86

5.15

2.81

87.3

3.44

2.0

ISO

2.43

 

 

4.43

3.9

91.6

CD54

3.67

1.24

112.7

3.92

CD86

5.55

3.12

96.9

3.35

2.4

ISO

2.44

 

 

4.16

3.7

95.7

CD54

3.54

1.10

100.0

3.77

CD86

4.95

2.51

78.0

3.27

2.9

ISO

2.50

 

 

4.17

3.8

94.4

CD54

3.62

1.12

101.8

3.78

CD86

5.03

2.53

78.6

3.40

3.5

ISO

2.55

 

 

4.19

3.7

95.7

CD54

3.61

1.06

96.4

3.74

CD86

5.15

2.60

80.7

3.27

4.2

ISO

2.68

 

 

4.38

3.9

91.5

CD54

3.84

1.16

105.5

3.84

CD86

5.53

2.85

88.5

3.49

5.0

ISO

2.83

 

 

5.14

4.5

80.1

CD54

4.43

1.60

145.5

4.69

CD86

6.30

3.47

107.8

3.55

 

Results of the second h-CLAT run for the Test Item Sodium cocopropylenediamine propionate, CAS 97659-50-2

 

Concentration (µg/mL)

Antibody / ISO

MFIGeoMean(FITC)

MFI-ISO

RFI (%)

Cyto(Geo)GeoMean(7-AAD)

Mean Cyto

Viability (%)

Medium control

-

ISO

2.02

 

 

4.09

3.9

100.0

CD54

3.23

1.21

100.0

3.80

CD86

4.46

2.44

100.0

3.68

DMSO control

-

ISO

2.05

 

 

4.15

3.8

100.0

CD54

3.36

1.31

100.0

3.60

CD86

4.48

2.43

100.0

3.55

Positive control (DNCB)

2

ISO

2.10

 

 

5.19

4.5

84.3

CD54

5.61

3.51

267.9

4.51

CD86

8.52

6.42

264.2

3.71

3

ISO

2.56

 

 

7.54

5.6

67.2

CD54

7.37

4.81

367.2

5.56

CD86

18.32

15.76

648.6

3.71

Test Item

1.4

ISO

2.09

 

 

4.12

3.8

100.5

CD54

3.30

1.21

100.0

3.76

CD86

4.13

2.04

83.6

3.63

1.7

ISO

2.10

 

 

4.11

3.7

103.3

CD54

3.46

1.36

112.4

3.58

CD86

4.39

2.29

93.9

3.51

2.0

ISO

2.17

 

 

4.35

3.9

100.1

CD54

3.42

1.25

103.3

3.69

CD86

4.27

2.10

86.1

3.52

2.4

ISO

2.14

 

 

4.50

4.0

95.6

CD54

3.40

1.26

104.1

3.95

CD86

4.17

2.03

83.2

3.65

2.9

ISO

2.31

 

 

4.67

4.2

91.2

CD54

3.75

1.44

119.0

4.08

CD86

4.40

2.09

85.7

3.93

3.5

ISO

2.37

 

 

4.62

4.1

94.1

CD54

3.81

1.44

119.0

4.01

CD86

4.54

2.17

88.9

3.67

4.2

ISO

2.46

 

 

4.70

4.4

88.3

CD54

4.38

1.92

158.7

4.27

CD86

5.50

3.04

124.6

4.14

5.0

ISO

2.68

 

 

5.59

5.1

75.7

CD54

4.95

2.27

187.6

4.88

CD86

6.22

3.54

145.1

4.82

 

Acceptance Criteria of the h-CLAT

Acceptance Criteria of the first h-CLAT run for the Test Item Sodium cocopropylenediamine propionate, CAS 97659-50-2

 

Cell viability of the DMSO control should be more than 90% in comparison to the medium control.

           DMSO/Medium = 102.9%

           Medium/DMSO = 97.2%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.

           2 µg/mL DNCB (CD 54):       250.0%

           2 µg/mL DNCB (CD 86):       332.9%

           3 µg/mL DNCB (CD 54):       445.6%

           3 µg/mL DNCB (CD 86):       519.4%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).

           CD54:   103.6%

           CD86:    89.8%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%.

           Medium CD 54:         151.4%

           Medium CD 86:         250.5%

           DMSO CD 54:           154.5%

           DMSO CD 86:           238.3%

Acceptance Criteria of the second h-CLAT run for the Test Item Sodium cocopropylenediamine propionate, CAS 97659-50-2

 

Cell viability of the DMSO control should be more than 90% in comparison to the medium control.

           DMSO/Medium =  97.7%

           Medium/DMSO = 102.4%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.

           2 µg/mL DNCB (CD 54):       267.9%

           2 µg/mL DNCB (CD 86):       264.2%

           3 µg/mL DNCB (CD 54):       367.2%

           3 µg/mL DNCB (CD 86):       648.6%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).

           CD54:   108.3%

           CD86:    99.6%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%.

           Medium CD 54:         159.9%

           Medium CD 86:         220.8%

           DMSO CD 54:           163.9%

           DMSO CD 86:           218.5%

 

Historical Control Data

These values represent historical control data of 66 runs conducted in 2015/2016.

Positive Control

DMSO- /Medium Control

DNCB (2 µg/mL)

DNCB (3 µg/mL)

(MFI - MFI(ISO)DMSO)/
(MFI - MFI(ISO)Medium)

Antibody

CD 54

CD 86

CD 54

CD 86

CD 54

CD 86

Value should be

≥ 200%

≥ 150%

≥ 200%

≥ 150%

< 200%

< 150%

Mean Value [%]

466.9

373.2

655.1

437.4

112.7

103.5

Standard Deviation

248.3

138.4

268.4

162.2

16.3

17.4

Max Value [%]

1714.1

837.1

1492.2

950.5

151.5

144.9

Low Value [%]

200.8

200.0

266.7

185.8

62.6

74.8

 

Interpretation of results:
other: The result is considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP)
Conclusions:
At test concentrations of up to 5 µg/mL, the test item Sodium cocopropylenediamine propionate, CAS 97659-50-2 did not activate THP-1 cells sufficiently to elicit a positive response under the test conditions of this study. Whilst the test item is regarded as a UVCB, this is due only to the presence of a fatty acid chain having a carbon chain distribution of C12 to C18 (predominantly C12 to C14). It is considered that the distribution of fatty acid chain length will not affect the validity of the h-CLAT result. Furthermore, although a Log Pow value of the test item is not available for the test item, it was possible to prepare suitable test concentrations in culture medium to ensure conduct of a valid h-CLAT. Therefore, the result is considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).
Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitisation AOP) of Sodiumcocopropylenediamine propionate, CAS 97659-50-2.

It was indicated by the Sponsor that whilst the test item is regarded as a UVCB, this is due only to the presence of a fatty acid chain having a carbon chain distribution of C12 to C18 (predominantly C12 to C14) and that this distribution of fatty acid chain length would not impact upon the validity of the h-CLAT result. Although a Log Pow value of the test item was not available for the test item, it was possible to prepare concentrations of test item in culture medium to ensure conduct of a valid h-CLAT. The highest test item concentration for the main h-CLAT experiment was previously determined by two valid and independent XTT tests. The h-CLAT was conducted by administration of the test item concentrations to THP-1 cells for 24 ± 0.5 hours.

Due to strong cytotoxicity observed in the first XTT test (dose finding assay, starting with 5000 µg/ml), the first XTT test was repeated with adjusted test item concentrations.

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 6.25 µg/mL up to the highest tested concentration (50 µg/mL) in both XTT tests (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 4.2 µg/mL.

The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT):

1.4, 1.7, 2.0, 2.4, 2.9, 3.5, 4.2 and 5 µg/mL

The test item was tested in 2 independent runs. Whilst a dose response was observed in both runs,the RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in both runs. Therefore the h-CLAT prediction is considered negative for the test item in this study.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria(CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

In conclusion, at test concentrations of up to 5 µg/mL, the test item Sodium cocopropylenediamine propionate, CAS 97659-50-2 did not activate THP-1 cells sufficiently to elicit a positive response under the test conditions of this study. Whilst the test item is regarded as a UVCB, this is due only to the presence of a fatty acid chain having a carbon chain distribution of C12 to C18 (predominantly C12 to C14). It is considered that the distribution of fatty acid chain length will not affect the validity of the h-CLAT result. Furthermore, although a Log Pow value of the test item is not available for the test item, it was possible to prepare suitable test concentrations in culture medium to ensure conduct of a valid h-CLAT. Therefore, the result is considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

During the development of this substance β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt originally tested asSodium cocopropylenediamine propionate, CAS 97659-50-2 it was tested in a guinea pig maximisation test. This gave a positive result but the sample was not typical of what is sold today. It had much higher levels of residual and by products so the result is considered Klimisch 3 and cannot be used to assess skin sensitisation for REACH. Three in-vitro studies, DPRA, KerationSens and h-CLAT have been performed on the current test substance which is imported as a ca. 30% solution in water. These are Klimisch 1 studies and are the basis of an assessment for skin sensitisation potential.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

The three in-vitro skin sensitisation studies have not shown any evidence of potential for skin sensitisation. In addition β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt CAS No 2136366-30-6, is sold as a ca. 30% solution in water. Inhalation is not an expected route of exposure. There are no standard animal models for respiratory sensitisation, so the lack of potential for skin sensitisation and lack of inhalation exposure support a conclusion that it should not be classified as a respiratory sensitiser.

Justification for classification or non-classification

During the development of this substance β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt originally tested as Sodium cocopropylenediamine propionate, CAS 97659-50-2 it was tested in a guinea pig maximisation test. This gave a positive result but the sample was not typical of what is sold today. It had much higher levels of residual and by products so the result is considered Klimisch 3 and cannot be used to assess skin sensitisation for REACH. Three in-vitro studies, DPRA, KerationSens and h-CLAT have been performed on the current test substance which is imported as a ca. 30% solution in water. These are Klimisch 1 studies and are the basis of an assessment for skin sensitisation potential.

The purpose of the DPRA study (based on the OECD guideline for the testing of chemicals,In chemicoSkin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of Sodium cocopropylenediamine proprionate (registered as β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt). 

Solutions of Sodium cocopropylenediamine proprionate were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall result of 2.79% depletion places Sodium cocopropylenediamine proprionate in the reactivity class of “no to minimal” and therefore it is predicted by DPRA to be a potential non-skin sensitizer

In the Keratinosens study the skin sensitisation potential was assessed by the induction of luciferase activity to>1,5 fold over the untreated controls. In this study during 3 runs this only occurred at a mid-concentration of 15.625µM in run 2 but there was no dose response. In the other two runs the highest induction was 1.275 in run one at 125µM and 1.375 in run 3 at 7.813µM. All the acceptance criteria were met and positive control responded correctly. The overall assessment was that the test substance was a non-sensitiser for human skin.

In the h-CLAT test report it was concluded that, at test concentrations of up to 5 μg/mL, the test item Sodium cocopropylenediamine propionate, CAS 97659-50-2 (registered asβ-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt )did not activate THP-1 cells sufficiently to elicit a positive response under the test conditions of this study. Whilst the test item is regarded as a UVCB, this is due only to the presence of a fatty acid chain having a carbon chain distribution of C12 to C18 (predominantly C12 to C14). It is considered that the distribution of fatty acid chain length will not affect the validity of the h-CLAT result. Furthermore, although a log Pow value for the test item was not available, it was possible to prepare suitable test concentrations in culture medium to ensure conduct of a valid h-CLAT. Therefore, the result is considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

All three in-vitro / in-chemico studies were negative, therefore based on this and lack of any structural indications of skin sensitisation potential β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt CAS No 2136366-30-6 is not classified for skin sensitisation.