Butyl toluene-4-sulphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 August 2019 to 16 August 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Butyl toluene-4-sulphonate

EC Number:

212-295-5

EC Name:

Butyl toluene-4-sulphonate

Cas Number:

778-28-9

Molecular formula:

C11H16O3S

IUPAC Name:

butyl 4-methylbenzene-1-sulfonate

Test material form:

liquid

Remarks:

(colourless to slightly yellow)

Details on test material:

- Appearance: Colourless to slightly yellow liquid
- Storage conditions: At room temperature protected from light

Specific details on test material used for the study:

No correction was made for the purity/composition of the test material.

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:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Source: MatTek Corporation (Ashland MA, USA)
- Tissue batch number(s): 30939 Kit G and Kit H
- Storage conditions: On the day of receipt, the tissues were kept on agarose and stored in the refrigerator.

TEST FOR DIRECT MTT REDUCTION
To assess the ability of the test material to reduce MTT, 50 μL of the test material or 50 μL Milli-Q water as a negative control were added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C. At the end of the exposure time it was checked if a blue/ purple colour change or a blue / purple precipitate was observed.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
To assess the colour interference, 50 μL of the test material or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C in the dark. At the end of the exposure time the mixture was shaken, and it was checked if a blue / purple colour change was observed.

MAIN TEST
PRE-INCUBATION
At least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 1 hour at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM just before the test material was applied.

APPLICATION
The liquid test material, positive and negative controls were applied undiluted (50 μL) directly on top of the tissue. To protect the test material from light amber coloured glassware was used or glassware was wrapped in tinfoil.

REMOVAL OF TEST MATERIAL AND CONTROLS
After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test material. 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.

CELL VIABILITY MEASUREMENTS
The DMEM was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37 °C in air containing 5 % CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol overnight at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

ENVIRONMENTAL CONDITIONS USED FOR TEST SYSTEM
All incubations, with the exception of the test material incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100 % (actual range 62 - 92 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.5 - 37.2 °C).

NUMBER OF REPLICATE TISSUES: The test was performed on a total of 4 tissues per test material together with a negative control and positive control. Two tissues were used for a 3-minute exposure to test material and two for a 1-hour exposure. 2 tissues were treated with negative control and 2 tissues were treated with positive control for both the 3-minute and 1-hour time point.

CALCULATION OF CELL VIABILITY
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc / mean ODlt_u+MTT) * 100

DATA EVALUATION
- Acceptability Criteria
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100 % viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30 %.

- Interpretation
A test material is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50 %.
b) In addition, a test material considered non-corrosive (viability ≥ 50 %) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test material is decreased below 15 %.
A test material is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50 %.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15 %.

- Data interpretation and sub-categorisation of test materials
< 50 % after 3-minute exposure = Corrosive
≥ 50 % after 3-minute exposure AND < 15 % after 1-hour exposure = Corrosive
≥ 50 % after 3-minute exposure AND ≥ 15 % after 1-hour exposure = Non-corrosive
For substances/mixtures identified as Corrosive:
< 25 % after 3-minute exposure = Sub-category 1A
≥ 25 % after 3-minute exposure = Sub-categories 1B and 1C

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution): 8N

Duration of treatment / exposure:

3 minutes, 1 hour

Duration of post-treatment incubation (if applicable):

3 hours with MTT

Number of replicates:

2 per time point

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application

Value:

107

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application

Value:

119

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DIRECT MTT REDUCTION
No blue / purple precipitate was observed. It was therefore concluded that the test material did not interact with MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
The solutions did not turn blue / purple. It was therefore concluded that the test material did not produce colour interference with the MTT endpoint.

MAIN STUDY
The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 107 and 119 %, respectively. As the mean relative tissue viability for the test material was not below 50 % after 3 minutes of treatment and not below 15 % after 1 hour of treatment, the test material 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 3-minute and 1-hour exposures to the positive control were 9.5 and 7.7 %, respectively.
- 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:

other: not corrosive according to EU criteria

Conclusions:

Under the conditions of the study, the test material was not corrosive.

Executive summary:

The in vitro skin corrosion potential of the test material was assessed in a study which was conducted in accordance with the standardised guidelines OECD 431 and EU Method B.40 BIS, under GLP conditions.

During the study, the human three-dimensional epidermal model (EpiDerm (EPI-200)) was used to evaluate the ability of the test material to induce skin corrosion. The possible corrosive potential of the test material was tested through topical application of 50 µL undiluted test material for 3 minutes and 1 hour.

Under the conditions of the study, the positive control had a mean relative tissue viability of 7.7 % after the 1-hour 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.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 107 and 119 %, respectively. As the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment, the test material is considered to be not corrosive.

In conclusion, the test material is not corrosive in the in vitro skin corrosion test under the experimental conditions described.