Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 27 September 2017 Experimental completion date: 13 October 2017
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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:
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
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

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 10 October 2017. On the day of experiment EpiSkin™ tissues were transferred to 12-well plates with maintenance medium and the pre-incubation phase of the EpiSkin™ tissues started.

EpiSkin™ Kit Components Needed for the Assay
EpiSkin™ Kit Lot No.: 17-EKIN-041
1, Sealed 12-well plate, Contains 12 inserts with EpiSkin™ tissues on agarose
1, 12-well plate, For MTT viability assay
1 bottle, Assay Medium, Basic medium for use in MTT assays
1 bottle, EpiSkin™ Maintenance Medium, Basic medium for incubations
Vehicle:
unchanged (no vehicle)
Details on test system:
Test for Direct MTT Reduction and Colour Interference
Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose the test item (10 μL) was mixed with 90 μL of deionised water in a pre-experiment. The test item/water mixture was gently shaken for 15 minutes at room temperature.
The colour of the test item/water mixture did not change during the incubation period compared with the colour of the pure test item.
For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability 10 μL of the test item was added to 2 mL of MTT solution (0.3 mg/mL) and the mixture will be incubated in the dark at 37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution was used as the control. Since the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.

Pre-warming of EpiSkin™ Tissues
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for about 22 hours.
Treatment
The negative control, positive control and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues for 15 minutes.
After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for about 42 at 37 ± 1.5 °C, 5 ± 0.5% CO2.
IL-1 α Immunoassay
Samples of all treatment groups were taken from the wells. The plates were shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well was taken and was stored in the freezer at –20 °C. Since the results derived from the MTT assay (see chapter 4.4 and 8.1) were not unclear or borderline, the IL-1 α concentration in the medium was not determined and the taken samples will be discarded after report finalization.
MTT Assay
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for 2.5 hours at room temperature with gentle agitation.
Per tissue sample 2  200 μL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Softmax Pro Enterprise, version 4.7.1) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.
Data Recording
The data generated were recorded in the laboratory protocol. The results were presented in tabular form, including experimental groups with the test item, negative and positive controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 μL (26 μL/cm2) of the undiluted test item were applied to each of the triplicate tissues.
10 μL of negative and positive were applied to each of triplicate tissues for 15 minutes.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
triplicate

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
100.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue/purple colour.
The mean relative absorbance value of the test item, corresponding to the cell viability, was 100.6% (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.
The acceptance criteria were met:
• the mean OD of the three negative control exposed tissues is ≥ 0.6 till ≤ 1.5 (range: 1.164 to 1.292).
• the standard deviations between tissues of the same treatment group was ≤ 18 (range: 3.0 to 5.5).
• the mean relative tissue viability of the positive control was ≤ 40% (22.1%).
• the acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (1.9 mg/mL).
• the results for the positive and negative controls are within the historical data (means, rel. standard deviation, and ranges) of Envigo CRS GmbH

Any other information on results incl. tables

Treatment Group

Tissue No.

OD 570 nm
Well 1

OD 570 nm
Well 2

Mean OD of 2 Wells

Mean OD

of 2 Wells blank

corrected

Mean

OD

of 3 tissues

blank corrected

Rel. Viability [%] Tissue
1, 2 + 3*

Standard Deviation

of viability values

Mean Rel. Viability

[%]**

Blank

 

0.037

0.040

0.039

0.000

 

 

 

 

Negative Control

1

1.306

1.278

1.292

1.253

1.184

105.9

5.5

100.0

2

1.209

1.214

1.211

1.173

99.1

3

1.176

1.152

1.164

1.125

95.1

Positive Control

1

0.263

0.255

0.259

0.221

0.261

18.6

3.0

22.1

2

0.312

0.318

0.315

0.277

23.4

3

0.324

0.325

0.324

0.286

24.2

Test Item

1

1.180

1.227

1.203

1.165

1.191

98.4

3.2

100.6

2

1.280

1.265

1.273

1.234

104.2

3

1.208

1.219

1.213

1.175

99.2

* Relative viability [rounded values]: [100 x (absorbance test item/ postive control/ negative control)]/ mean absorbance negative control

** Mean relative viability [rounded values]: [100 x (mean absorbance test item/ postive control/ negative control)]/ mean absorbance negative control

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, Sodium cocopropylenediamine propionate, CAS 97659-50-2 is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of Sodium cocopropylenediamine propionate, CAS 97659-50-2 by means of the Human Skin Model Test according to OECD TG 439.

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not dye water, when mixed with it (pre-test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative control (PBS) or the positive control (5% sodium lauryl sulfate) for 15 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD  0.6 till ≤ 1.5 thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

After treatment with the test item the mean relative absorbance value was 100.6%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Sodium cocopropylenediamine propionate, CAS 97659-50-2 is not irritant to skin according to UN GHS and EU CLP regulation.