Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 02, 2017 to October 06, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU B.40 BIS "In Vitro Skin Corrosion: Human Skin Model Test
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Chemical name (IUPAC), synonym or trade name: Phosphoric acid, mono-and di-C8-10-(even numbered)-alkyl esters
CAS number: 98653-76-0
Molecular structure: UVCB
Molecular formula: UVCB

Specific details on test material used for the study:
- Name of test material (as cited in study report): Phosphoric acid, mono-and di-C8-C10-(even numbered)-alkyl esters
- Batch: CH 205624/001
- Composition: UVCB
- Appearance: Clear colourless liquid
-Test item storage: At room temperature
-Stable under storage conditions until: 01 July 2018 (expiry date)

In vitro test system

Test system:
human skin model
Remarks:
EpiDerm Skin Model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The model 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 EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts. Recommended test system in international guidelines (OECD and EC)
Vehicle:
unchanged (no vehicle)
Details on test system:
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 h before the assay was started the tissues were transferred to 6-well plates containing 0.9 ml DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 3 h at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before the test substance was applied. The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test substance and two for a 1-h exposure. 50 µL of the undiluted test substance was added into the 6-well plates on top of the skin tissues. For the negative and positive controls, 2 tissues were treated with 50 µl Milli-Q water (negative control) and 2 tissues were treated with 50 µl 8N KOH (positive control) for both the 3-minute and 1-h time point. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test substance. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µl DMEM until 6 tissues (= one application time) were dosed and rinsed.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 µL
Duration of treatment / exposure:
3 minutes and 1 h
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
cytotoxicity
Run / experiment:
3 minutes treatment
Value:
86
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
cytotoxicity
Run / experiment:
1 h treatment
Value:
9.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- The test substance was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test substance did not interfere with the MTT endpoint.

- The mean absorption at 570 nm measured after treatment with the test substance and controls were as follows:
1) Negative control 1.988 ± 0.253 (3 minute application) and 1.857 ± 0.050 (1 h application)
2) Test substance 1.710 ± 0.127 (3 minute application) and 0.170 ± 0.023 (1 h application)
3) Positive control 0.333 ± 0.247 (3 minute application) and 0.137 ± 0.028 (1 h application)

- The mean tissue viability obtained after 3 minute and 1 h treatments with the test substance compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after the 3 minute and 1 h treatments with the test substance compared to the negative control tissues was 86% and 9.1% respectively. Because the mean relative tissue viability for the test substance was below 15% after 1 hour treatment it is considered to be corrosive.

- The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1h exposure to the positive control was 7.4%.

- In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 16%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
Under the study conditions, the substance was found to be corrosive to human skin.
Executive summary:

A study was conducted to determine the in vitro skin corrosion potential of the substance according to OECD Guideline 431and EU Method B.40 bis, in compliance with GLP. Duplicate tissue samples were exposed to 50 µL test substance for 3 minutes and 1 h. After the treatment period, a determination of the cytotoxic effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin corrosion was expressed as the remaining cell viability after exposure to the test substance. The positive control showed a mean relative tissue viability of 7.4% after 1 h exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (≥0.8 and ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the coefficient of variation between tissue replicates was ≤16%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 3 minute and 1 h treatments with the test substance compared to the negative control tissues was 86% and 9.1%, respectively. Because the mean relative tissue viability for the test substance was below 15% after the 1 h treatment it is considered to be corrosive to the skin. Under the study conditions, the substance was found to be corrosive to human skin (Groot, 2017).