Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May - Aug 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt Rheinland-Pfalz

Test material

Constituent 1
Chemical structure
Reference substance name:
Dibenzyldimethylammonium chloride
EC Number:
202-903-7
EC Name:
Dibenzyldimethylammonium chloride
Cas Number:
100-94-7
Molecular formula:
C16H20N.Cl
IUPAC Name:
dibenzyldimethylazanium chloride
Test material form:
solid: crystalline
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 17 Jan 2020
- Physical state / color: Solid / white
- pH value: ca. 5 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, under N2

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For better handling, the test substance was ground with a pestle and mortar.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The test is based on the experience that corrosive and irritant chemicals produce cytotoxicity in human reconstructed epidermis after a short term topical exposure. The test is designed to predict a skin corroion or irritation potential of a chemical by using the three-dimensional human epidermis
model EpiDerm. After application of the test material to the stratum corneum surface of the EpiDerm tissue, the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow-colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue-colored formazan. After isopropanol extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. The optical density of the extracts of tissues treated with the test substance is compared to negative control values from tissues and expressed as relative tissue viability.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number(s): 28696
- Date of initiation of testing: 10.05.2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 28 min at room temperature and 32 min in the incubator
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: one washing step
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mL MTT solution
- Incubation time: 3 h
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm without reference filter
- The test substance is not able to reduce MTT directly, which was demonstrated in a pretest. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was not introduced.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze-killed
- N. of replicates: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-irritant to skin if the viability after exposure and post-treatment incubation is greater than 50 %
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL bulk volume (about 19 mg)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 % in water
Duration of treatment / exposure:
1 h
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st test run
Value:
96.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2nd test run
Value:
94.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3rd test run
Value:
76
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Table 2: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficients of variation

Test substance identification

 

Tissue 1

Tissue 2

Tissue 3

Mean

SD

CV [%]

NC

Mean OD570

1.687

1.812

1.687

1.729

 

 

Viability

[% of NC]

97.6

104.8

97.6

100.0

4.2

4.2

Test substance

Mean OD570

1.661

1.638

1.315

1.538

 

 

Viability

[% of NC]

96.1

94.8

76.0

89.0

11.2

12.6

PC

Mean OD570

0.059

0.061

0.068

0.062

 

 

Viability

[% of NC]

3.4

3.5

3.9

3.6

0.3

7.3

Table 3: Historic control data of NC and PC of skin irritation test

Historic Range of NC

OD570

 

 

 

 

Period

Mean OD

SD

Mean + 2 SD

Mean – 2 SD

Jan 2017 – Mar 2019

1.831

0.192

2.215

1.447

Historic Range of PC

OD570

 

 

 

 

Period

Mean OD

SD

Mean + 2 SD

Mean – 2 SD

Jan 2017 – Mar 2019

0.048

0.005

0.059

0.037

Relative Viability [%]

Period

Mean %

SD

Mean + 2 SD

Mean – 2 SD

Jan 2017 – Mar 2019

2.6

0.4

3.4

1.9

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm in vitro skin irritation and corrosion test strategy under the test conditions chosen.
Executive summary:

The objective of the study was to assess the skin irritation and corrosion potential of the test substance. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

However, in the current case the results derived with SIT alone were sufficient for a final assessment. Therefore, further testing in SCT was waived.

The potential of the test substance to cause dermal irritation was assessed by a single topical application of ca. 25 μL bulk volume (about 19 mg) undiluted ground test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). Before application the epidermis surface was moistend with 25 μL sterile PBS. Thereafter, the solid ground test material was applied directly onto the tissue surface and distributed together with the fluid, so that the surface of the epidermis model was uniformly and completely covered.

The Skin Irritation Test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. In addition to the test substance, 30 μL / tissue of a negative control (PBS) and a positive control (5% SDS) were applied to three tissues each.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The following results were obtained in the EpiDerm™ Skin Irritation Test (SIT):

The test substance is not able to reduce MTT directly, which was demonstrated in a pretest. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was not introduced.

The relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 89%.

Application of the positive control 5% SDS showed a relative mean viability of the tissues of 3.6% and reflects the expected sensitivity of the tissues. The mean OD570 of the negative control (PBS) fulfill the acceptance criteria and demonstrate the validity of the assay.

Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.