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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-12-15 to 2016-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted July 28, 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(hexadecylamidopropyl)trimethylammonium chloride
EC Number:
257-104-6
EC Name:
(hexadecylamidopropyl)trimethylammonium chloride
Cas Number:
51277-96-4
Molecular formula:
C22H47N2O.Cl
IUPAC Name:
(3-hexadecanamidopropyl)trimethylazanium chloride
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
recommended by guideline
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEMEpiSkin™ Kit Lot No.: 16-EKIN-002, SkinEthic Laboratories (69007 Lyon, France)EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes 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.The tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS on 12 January 2016. On day of experiment EpiSkin™ tissues were transferred to 12-well plates with maintenance medium and the preincubation phase of the EpiSkin™ tissues started.REMOVAL OF TEST SUBSTANCEAfter the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle 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.MTT ASSAY:The MTT concentrate was prepared freshly. A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.After the treatment procedure 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 aspirated from the wells and the wells were rinsed three times with PBS. 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 vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted 69 hours and 15 min without shaking in the refrigerator.Per each tissue sample 2 × 200 μL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader with 570 ± 1 nm filter. Mean values were calculated from the 2 wells per tissue sample.PREDICTION MODEL / DECISION CRITERIA A test substance is considered irritant in the skin irritation test if:The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.A test substance is considered non-irritant in the in vitro skin irritation test if:The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 5 μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 10 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.NEGATIVE CONTROL- Amount(s) applied (volume or weight): 10 µL deionised waterPOSITIVE CONTROL- Amount(s) applied (volume or weight): 10 μL 5% SDS
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
triplicates

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test substance
Value:
98
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
32.7% tissue viability
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. In a further pre-test the test item was not a MTT reducer

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline:
Positive Control range of viability: 1.7% - 35.4% (19.3% ± 10.0%; n = 232)
Negative Control range of viability (OD): 0.607 – 1.521 (1.004 ± 0.219; n = 232)

Any other information on results incl. tables

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Group

Absorbance

570 nm

Tissue

1

Absorbance

570 nm

Tissue

2

Absorbance

570 nm

Tissue

3

Mean

Absorbance

of

3

Tissues

Relative

Standard

Deviation

[%]

Rel. Absorbance

[% of

Negative

Control]

Negative

Control

0.872

0.981

0.915

0.922

 

6.0

100.0

Positive

Control

0.307

0.313

0.286

0.302

4.7

32.7

Test item

0.941

0.794

0.976

0.904

10.7

98.0

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
C16 Alkylamidopropyltrimethylammonium Chloride (100% a.i.) was not irritating in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015), C16 Alkylamidopropyltrimethylammonium Chloride (100% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 15 minutes in triplicates. 5 μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 10 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

After 15 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (deionised water) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment with C16 Alkylamidopropyltrimethylammonium Chloride compared to the negative control tissues was 98%. Since the mean relative tissue viability for the test substance was above 50%, C16 Alkylamidopropyltrimethylammonium Chloride is identified to be not irritating.