Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzyltris(dimethylaminato)phosphorus(1+) tetrafluoroborate(1-)
EC Number:
302-080-5
EC Name:
Benzyltris(dimethylaminato)phosphorus(1+) tetrafluoroborate(1-)
Cas Number:
94088-77-4
Molecular formula:
C13H25BF4N3P
IUPAC Name:
benzyltris(dimethylamino)phosphanium; tetrafluoroboranuide
Test material form:
solid: crystalline

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: human-derived epidermal keratinocytes
Details on test system:
The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, 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 representing main
lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.

EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratisla-va.
Designation of the kit: EPI-200-SIT
Day of delivery: 06. Sep. 2016
Batch no.: 23353
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25.1 mg, 24.8 mg, 24.9 mg

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL 5% SDS-solution
Duration of treatment / exposure:
Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates are trans-ferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were set in the incubator for 23 hours and 29 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.
Duration of post-treatment incubation (if applicable):
The tissues were removed from the incubator and shaken for 5 minutes (120 rpm). After that, 0.9 mL assay medium were filled in the lower row of the 6-well-plate Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 hours and 25 minutes for post-incubation at 37 ± 1°C and 5.0 ± 0.5% CO2.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: Optical Density (Mean absorbance values)
Run / experiment:
1-3
Value:
1.68
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

 Measured Values

As blank, the optical density of isopropanol was measured in 8 wells of the 96-well-plate. The measured values and their mean are given in the following table:

Table9.1‑a      Absorbance values blank isopropanol (OD 570 nm)

Replicate

1

2

3

4

5

6

7

8

Mean

Absorbance

0.047

0.043

0.043

0.042

0.040

0.039

0.040

0.037

0.041

 

The absorbance values of negative control, test item and positive control are given in the following table:

Table9.1‑b      Absorbance Values negative control, test item and positive control (OD 570 nm)

Designation

Measurement

Negative Control

Tecnoflon BA 104

Positive Control

Tissue 1 

1

2.078

1.656

0.115

2

2.040

1.668

0.112

Tissue 2 

1

2.082

1.681

0.111

2

2.091

1.678

0.112

Tissue 3 

1

2.013

1.838

0.121

2

2.039

1.804

0.118

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in table 9.1-a. The mean of the three tissues was also calculated.

Table9.1‑c      Mean Absorbance Values

Designation

Negative Control

Tecnoflon BA 104

Positive Control

Mean – blank (tissue 1)

2.018

1.621

0.073

Mean – blank (tissue 2)

2.046

1.639

0.071

Mean – blank (tissue 3) 

1.985

1.780

0.079

Mean of the three tissues

2.016

1.680

0.074

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Three tissues of the human skin model EpiDermTM were treated with Tecnoflon BA 104 for 60 minutes.
The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).
DPBS-buffer was used as negative control, 5% SDS solution was used as positive con-trol.
After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 2.0. The positive control showed clear irritating effects. Relative absorbance was reduced to 3.7% (required: < 20%).
Variation within the tissue replicates was acceptable (required: ≤ 18%).
After the treatment with the test item, the relative absorbance values were reduced to 83.3 %. This value is above the threshold for skin irritation potential (50%).
Therefore, Tecnoflon BA 104 is considered as non-irritant to skin in the Reconstructed Human Epidermis (RhE) Test Method.
Executive summary:

The test item is considered as non-irritant to skin.

After the treatment, the relative absorbance values were reduced to 83.3%. This value is above the threshold for skin irritation (50%).

The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8.

The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system.

Variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18%).

For these reasons, the result of the test is considered valid.