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Skin irritation / corrosion

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skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date :November 8, 2017 - Experimental completion date : November 10, 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 22, 2010
GLP compliance:
Ashland testing laboratory works with Standard Operating Procedures.

Test material

Constituent 1
Chemical structure
Reference substance name:
1-Propanaminium, 3-amino-N-(2-hydroxyethyl)-N,N-dimethyl-, N-mink-oil acyl derivs., chlorides
EC Number:
EC Name:
1-Propanaminium, 3-amino-N-(2-hydroxyethyl)-N,N-dimethyl-, N-mink-oil acyl derivs., chlorides
Cas Number:
Molecular formula:
UVCB: CnHnN2 O2 (Cl)
1-Propanaminium, 3-amino-N-(2-hydroxyethyl)-N,N-dimethyl-, N-mink-oil acyl derivs., chlorides
Specific details on test material used for the study:
Chemical name: CERAPHYL™ 65
Code 826812
Batch number 0001954899
Supplier Ashland
Dry matter (%) 59.4%
Physical state Clear amber to dark amber liquid
pH 7.6
Solvent Propylene Glycol
Treatment None
Storage conditions Room Temperature

In vitro test system

Test system:
human skin model
Source species:
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
unchanged (no vehicle)
Details on test system:
- Ashland 0.5 cm2 reconstructed epidermis from normal human keratinocytes.
- Batch N°: 1710EPID02
- Origin: Foreskin
- Quality Controls: Certified by Ashland Tissue Engineering laboratories, 06/11/2017
- Cells were grown on inert polycarbonate filter on chemically defined medium, airlifted for 17 days
- 3 tissues were used for each chemical.
- The RHE tissues were incubated at 37°C, 5% CO2 until test substance application (usually 24 hours).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Duration of treatment / exposure:
42 minutes
. Fill a 24-well plate with 300μl by well of pre-warmed maintenance culture medium.
· Transfer tissues.
· Dispense 16 μL ± 0.5 μL (or 16 μg) of products and controls on the top of epidermis.
· Carefully apply a nylon mesh (Ø = 7.5mm) on the whole surface with forceps.
· Keep the plates containing the treated epidermis for 42 minutes at room temperature.
Duration of post-treatment incubation (if applicable):
42 hours and MTT incubation for 3 hours.
· Remove the nylon mesh with fine forceps from the epidermal surface of a treated tissue.
· Remove the treated units using forceps, and rinse thoroughly 25 times with 1 ml DPBS.
· Transfer the washed tissue on 2 mL growth culture medium (6-well plate).
· Incubate the treated and rinsed epidermis at 37°C, 5% CO2 for 42 hours (± 60 minutes).
Assessment of non-specific MTT reduction (NSMTT)
· To identify direct MTT reducers, each test chemical should be added to freshly prepared MTT solution. If the MTT mixture containing the test chemical turns blue/purple, the test chemical is presumed to directly reduce MTT and a further functional check on non-viable RhE tissues should be performed.
· Each MTT reducing test chemical is applied on at least two killed tissue replicates which undergo the entire testing procedure to generate a non-specific MTT reduction (NSMTT)
Number of replicates:
3 per group: Negative control, Quaternium-26, Positive control

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Executive summary:

According to ASHLAND EQUIVALENT EPIDERMISES SKIN IRRITATION test, CERAPHYL™ 65 is classified as Non irritant.