Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

In vitro skin corrosion test with EpiDerm (EPI-200))

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:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU B.40 BIS "In Vitro Skin Corrosion: Human Skin Model Test

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol]

Molecular formula:

UVCB

IUPAC Name:

Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol]

Test material form:

liquid

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 ± 1.0˚C. 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

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

cytotoxicity

Run / experiment:

3 minutes treatment

Value:

ca. 71

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

cytotoxicity

Run / experiment:

1 h treatment

Value:

ca. 103

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

-The test substance was checked for color 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 1.407 ± 0.364 (3 minute application) and 1.904 ± 0.197 (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 71% and 103% respectively. Because the mean relative tissue viability for the test substance was not below 50% after 3 minutes treatment and not below 15% after 1 h treatment the test substance is considered to be not 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 1 h 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. The coefficient of variation between tissue replicates treated with the test substance was 31% for the 3-minute treatment, which is above acceptance criteria. Since all individual viabilities were >50%, the test outcome was considered valid.

Applicant's summary and conclusion

Interpretation of results:

other: CLP criteria not met

Conclusions:

Under the study conditions, the test substance was determined to be non corrosive to skin based on human three dimensional epidermal model (EpiDerm (EPI-200)).

Executive summary:

An in vitro study was conducted to determine the skin corrosion potential of the test substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], according to OECD Guideline 431 and EU Method B.40 bis, in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible interference with MTT reduction by adding 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. Duplicate tissue samples were exposed to 50 µL test substance for 3 min 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 potassium hydroxide showed a mean relative tissue viability of 7.4% after the 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 (lower acceptance limit ≥0.8 and upper acceptance limit ≤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 min and 1 h treatments with the test substance compared to the negative control tissues was 71% and 103%, respectively. Because the mean relative tissue viability for the test substance was above 50 and 15% after 3 min and 1 h treatment respectively, the test substance is considered to be non- corrosive. Under the study conditions, the test substance was determined to be non-corrosive to skin based on human three dimensional epidermal model (EpiDerm 200) (Groot, 2017).