Quaternary ammonium compounds, C12-14-alkyltrimethyl, Me sulfates

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 July 26, 2017 to July 27, 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, C12-14-alkyltrimethyl, Me sulfates

EC Number:

306-238-4

EC Name:

Quaternary ammonium compounds, C12-14-alkyltrimethyl, Me sulfates

Cas Number:

96690-44-7

Molecular formula:

R-N(Me)3+ CH3SO4- Where R = C12-14 alkyl chain

IUPAC Name:

dodecyltrimethylazanium trimethyl(tetradecyl)azanium dimethyl sulfate

Test material form:

solid

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:

Test system specifications: Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum was supplied by MatTek In Vitro Life ScienceLaboratories, Bratislava, Slovak Republic.

System conditions: Human keratinocytes are used to construct the epithelium. Multiple layers of viable epithelial cells are present under a functional stratum corneum. The stratum corneum is multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevent the passage of material around the stratum corneum to the viable model tissue. The skin model was supplied free of contamination with bacteria, mycoplasma and fungi.

Assessment of MTT Interacting substances:In order to assess the potential non-specific reduction of the test article, 25 mg of test substance was added to 1 mL of 1.0 mg/mL MTT and the colour change was assessed after incubation for 60 minutes at 37±1°C, 5±1% CO2, 95% RH. There was no change in colour therefore the test article did not interact with MTT.

Assessment for colour interference: 25 mg of test substance was added to both 0.3 mL deionized water and isopropanol before being incubated for 60 minutes at 37±1°C, 5±1% CO2, 95% RH. The solution did not become coloured, therefore the test article was deemed not to have the potential to stain the tissue.

Application of test and control substances: On the day of receipt EpiDerm tissues were transferred to refrigerator at 2 to 8°C and stored overnight. The next day, approximately 1.5 hours before starting the assay, the tissues were prepared for treatment in labelled 6-well plates. The test was performed on a total of four tissues per test substance, negative and positive control, out of which two were used for a 3 minute application and two tissues were used for a 1 hour application. Approximately 25 mg of the test substance was applied to the tissue, which was pre-moistened with 25 μL water to ensure good contact with the tissue surface. Further tissues were concurrently treated with 50 μL distilled water (negative control) and with 50 μL 8M potassium hydroxide (positive control). After the 3 minute or 1-hour contact periods, the tissues were washed with phosphate buffered saline (PBS) to remove residual material. The rinsed tissues were kept in 24-well plates (holding plates) until all tissues were dosed and rinsed. The positive control material was a direct MTT reducer, therefore additional positive controls were performed. The positive control article was applied to two freeze-killed tissues (thawed on the day of treatment) per exposure time. Cell viability measurements: 1 mg/mL MTT-medium and were incubated for 3 hours (37±1°C, 5±1% CO2, 95% RH). After incubation any resultant colour was extracted with 2 mL isopropanol overnight. The optical density of each resultant extract was determined spectrophotometrically at 570 nm and cell viability was calculated for each tissue as a percentage of the mean of the negative control tissue. Skin corrosivity potential of the test substance was classified according to the remaining cell viability obtained after test substance treatment with either of the two treatment times.

Cell viability measurements: Once all tissues had been rinsed, they were transferred to wells containing 300 μL of 1 mg/mL MTT-medium and were incubated for 3 hours (37±1°C, 5±1% CO2, 95% RH). After incubation any resultant colour was extracted with 2 mL isopropanol overnight. The optical density of each resultant extract was determined spectrophotometrically at 570 nm and cell viability was calculated for each tissue as a percentage of the mean of the negative control tissue. Skin corrosivity potential of the test article was classified according to the remaining cell viability obtained after test article treatment with either of the two treatment times.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg pre-moistened with 25 μL water to ensure good contact with the tissue surface

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 min treatment

Value:

ca. 58

Vehicle controls validity:

valid

Negative controls validity:

not 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. 20.4

Vehicle controls validity:

valid

Negative controls validity:

not 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 1.103 (3 minute application) and 1.115 (1 h application)
2) Test substance 0.634 (3 minute application) and 0.228 (1 h application)
3) Positive control 0.312 (3 minute application) and 0.186 (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 58% and 20.4% 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 OD values for the negative controls were between 0.8 and 2.8, and were within observed historical ranges. The mean relative tissue viability following the 1 h exposure to the positive control was -0.4%.


- In the range of 20 - 100% viability the coefficient of variation between tissue replicates was <30%, indicating that the test system functioned properly. The coefficient of variation between tissue replicates treated with the test substance was 29.97% 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:

A study was conducted to determine the in vitro skin corrosion potential of the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, 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 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. Duplicate tissue samples were exposed to approximately 25 mg of the test substance, which was pre-moistened with 25 μL water to ensure good contact with the tissue surface. Post treatment, 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 had a mean relative tissue viability of -0.4% 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 Guideline 431 and the laboratory historical control data range. In the range of 20 - 100% viability the coefficient of variation between tissue replicates was <30%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 3 min and 1 h treatment periods with the test substance compared to the negative control tissues were 58% and 20.4%, respectively. Because the mean relative tissue viability for the test substance was above 50 and 15% after 3 min and 1 h respectively, so 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 (Payne, 2017).