2-methylpropane-2-thiol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 January 2020 - 23 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

14 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-methylpropane-2-thiol (CAS # 75-66-1)

IUPAC Name:

2-methylpropane-2-thiol (CAS # 75-66-1)

Details on test material:

TS: 2-methyl-2-propanethiol
Purity 99.0-99.9%
Lot No. 200501-001

Specific details on test material used for the study:

Name: Tertiary Butyl Mercaptan

Synonyms:
- t-Butyl Mercaptan
- tert-Butanethiol
- 2-Methyl Propane-2-Thiol
- Tertiary Butyl Mercaptan (TBM)
- TERT BUTYL MERCAPTAN

CAS No.: 75-66-1
Batch No.: 2019-10-1008
Description: Clear liquid
Storage conditions: At room temperature
Purity: 99.94 wt %
Expiry (or re-test) date: 08 October 2021

Test animals / tissue source

Species:

other: reconstructed human Cornea-like epithelium (tissues)

Details on test animals or tissues and environmental conditions:

Cell type: non-transformed keratinocytes
Cell supplier: MatTek, Bratislava, Slovak Republic

Details on test system and justification for its use:
The EpiOcularTM model consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a nonkeratinized epithelium which models the cornea epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotypic 3D cornea-like model. The 3D tissue consists of highly organized cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration: neat (as supplied),
- Amount applied: 50 µL per tissue, taking care to spread it over the whole tissue surface area without damaging the tissue samples.

Duration of treatment / exposure:

Exposure period of 30 minutes (± 2 minutes) at +37°C, 5% CO2 in a humidified incubator, followed by rinsing.

Duration of post- treatment incubation (in vitro):

Post-soak period of 12 minutes (± 2 minutes) at room temperature.
Post-incubation period of 2 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator.

Number of animals or in vitro replicates:

The test item and both negative and positive controls were applied on duplicate tissues.
At the end of the viability assay, formazan level in tissues was assessed in duplicate for each tissue.
The mean OD values were then calculated from the four replicates of each item.

Details on study design:

PRELIMINARY TESTS
* Test for Direct MTT Reduction with the Test Item
As the test item may directly reduce MTT, thus mimicking mitochondrial succinate dehydrogenase activity, it is necessary to test its ability to directly reduce MTT before performing the main test. To identify any test substance interference with the MTT endpoint, the following preliminary test was performed:
- 50 µL of the test item were added to 1 mL of a 1.0 mg/mL freshly prepared MTT solution,
- a negative control was tested concurrently by adding 50 µL of sterile deionized water to 1 mL of a 1.0 mg/mL freshly prepared MTT solution,
- a sealer was placed on each plate before putting the plastic lids back, to avoid evaporation and/or cross-contamination,
- both mixtures were incubated in darkness at +37°C for 3 hours (± 10 minutes).
Then the colour of both solutions obtained was evaluated. If the MTT solution colour turns blue/purple when compared to the negative control, the test item was presumed to reduce MTT and additional controls were performed on freeze-dead tissues in parallel to the main test, to evaluate the part of OD due to the non-specific reduction of the MTT. Otherwise, no additional tissue controls were used.
 
* Test for the Detection of the Colouring Potential of the Test Item
As a test item may be coloured or become coloured in contact with water or isopropanol, it is necessary to test its potential interference with the MTT determination.
The ability of the test item to absorb significantly light at the wavelength used for MTT determination was tested as follows: a volume of 50 µL of the test item was added to 1 mL of water and a sealer was placed on each plate before putting the plastic lids back, to avoid evaporation and/or cross-contamination. Plate was then incubated for at least 1 hour at +37°C, 5% CO2.
Then, two 200 µL aliquots of test item solution and water (blank) were transferred to a 96-well plate and absorbance was measured.
If, after subtraction of the blank OD, the OD of the test item solution was > 0.08 (approximately 5% of the mean viability of the negative control) the test item was considered as having colouring potential and additional controls were performed on viable tissues in parallel to the main test. Otherwise, no additional tissue controls were used.

MAIN TEST
One 6-well plate was used for each item: one for tissues treated with the test item, one for tissues treated with the positive control and another one for those treated with the negative control. Each item was applied on duplicate tissues.

In addition, as the test item was found to have direct MTT reducing properties in the preliminary test, additional controls (i.e. two freeze-dead tissues treated with the test item and two freeze dead tissues treated with the negative control) were added in the main test for the evaluation of the OD part due to non specific MTT reduction. One 6-well plate was used for each of these control types.
Freeze-dead tissues have no metabolic activity, but absorb and bind the test item in the same way as viable tissues. It should be noted that freeze-dead tissues show a small amount of MTT reduction due to residual reducing enzymes within the tissue.
Freeze-dead tissues were prepared by placing untreated EpiOcularTM tissues in a 24 well plate. The tissues were then placed in a freezer (-20°C) overnight, thawing to room temperature, and then refreezing. Once frozen, the tissues were stored indefinitely in the freezer.

PRE-INCUBATION OF THE TISSUES
As the tissues were shipped the day prior the treatment, tissues were stored between +2°C and +8°C, prior their pre-incubation. The tissues were then equilibrated (in the 24-well shipping container) to room temperature for at least 15 minutes. Each tissue was inspected as specified in internal procedure, removed from the 24-well shipping containers, placed in a 6-well plate containing 1 mL of pre-warmed assay medium and then incubated for 1 hour (± 5 minutes) at +37°C, 5% CO2 in a humidified incubator.
After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24h) at +37°C, 5% CO2 in a humidified incubator. Each 6-well plate was labeled with the test item or control codes.

TREATMENT
Following the pre-incubation period, the tissues were pre-wetted with 20 µL of D-PBS, inserts were tapped to ensure that the entire tissue surface was wetted and plates were then incubated at +37°C, 5% CO2 in a humidified incubator for 30 minutes (± 2 minutes).
The test item, the negative control and the positive control were applied topically on each designated tissue, and gently spread onto the epithelium surfaces to ensure uniform covering of the tissues. Inserts were tapped on the wall/well of the plate to ensure that each item was correctly spread across the entire surface of each tissue.
All tissues (test item, negative and positive controls) were incubated at +37°C, 5% CO2 in a humidified incubator for 30 minutes (± 2 minutes).
The tissues were processed (pre-wetting, treatment and rinsing) in the same order and at regular time intervals to ensure each tissue receives an equal exposure period. In addition, during pre wetting and treatment, a sealer was placed on each plate before putting the plastic lids back and incubating in the incubator, to avoid evaporation and/or cross-contamination.

RINSING
At the end of the treatment period, each tissue was removed from the well of the treatment plate and rinsed to gently remove any residual test or control items. A set of three clean beakers containing a minimum of 100 mL each of D-PBS was used per test or control item. Items were first removed from the tissue surface by tapping upside down each insert onto a clean absorbent paper. Tissues were then dipped into the first beaker of D-PBS, swirled in a circular motion during approximately 2 seconds, lifted out and decanted back into the beaker. This process was performed three times per beaker. Any remaining liquid was decanted onto an absorbent paper.

POST-SOAK AND POST-INCUBATION
The rinsed tissues were transferred to new wells of a pre-labeled 12-well plate containing 5 mL of assay medium pre-warmed at room temperature and were then incubated for 12 minutes (± 2 minutes) at room temperature, in order to remove any items from the tissue.
At the end of the post-soak immersion, each insert was blotted on absorbent material and transferred to appropriate well of the pre-labeled 6-well plate containing 1 mL of assay medium. A sealer was placed on each plate before putting the plastic lids back, to avoid evaporation and/or cross-contamination. Then, tissues were incubated for 2 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator.

MTT VIABILITY ASSAY
Following the post-treatment incubation, a volume of 0.3 mL of a freshly prepared MTT solution at 1.0 mg/mL was added into new wells of pre-labeled 24 well plates.
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent paper. Tissues were then transferred to the MTT pre-filled wells, a sealer was placed on each plate before putting the plastic lids back, to avoid evaporation and/or cross-contamination. Then, tissues were incubated for 3 hours (± 10 minutes) at +37°C, 5% CO2 in a humidified incubator.
At the end of the 3-hour incubation period, the underside of each tissue was blotted on absorbent paper to dry. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated.
As the test item was a non-colourant liquid, each insert of tissues was transferred to new wells of the 24 well plate containing 2 mL of isopropanol per well so that isopropanol was flowing into the insert on the tissue surface. Plates were surrounded with parafilm to prevent evaporation. Formazan extraction was performed overnight at +2-8°C and protected from light.

OPTICAL DENSITY MEASUREMENTS
At the end of the formazan extraction period, the plates were placed under orbital shaking at room temperature for 15 minutes prior using them. Then, all tissues (treated with the test item, the negative control and the positive control) were pierced.
The extract solution was mixed using a pipette and two 200 µL aliquots were transferred to the appropriate wells of a pre-labeled 96-well plate. One 96-well plate was used for the negative and positive controls (placed at opposite end of the plate), and a separate 96-well plate was used for all test item-treated tissues (including additional freeze-dead). As the test item was found to have MTT reducing properties in the preliminary assay, another 96-well plate was also used for the negative control-treated freeze-dead tissues.
For each 96-well plate, the average Optical Density value (OD) of four wells containing 200 µL of isopropanol only was used as the blank. The OD was measured at 570 nm using a plate reader.

DATA ANALYIS
On each plate, the mean blank OD value (mean ODblank) was calculated from the four replicates. Then, the mean ODblank was subtracted from each OD value and the corrected mean OD values (mean cOD) of the two aliquots were calculated for each tissue. The mean cOD of the two negative control-treated tissues (mean cODNC) was set to 100% viability and was used as reference.
For the tissues treated with the test item and the positive control, the relative viability of each tissue was expressed as percentage of the reference viability and were calculated as follows:
Relative viability (%) = (cODTI or PC / mean cODNC) x 100

With:
cODTI = corrected OD of each tissue treated with the test item,
cODNC = corrected OD of each tissue treated with the negative control,
cODPC = corrected OD of each tissue treated with the positive control.

As the test item was found to have direct MTT reducing properties in the preliminary test, two freeze-dead tissues treated with the test item and two freeze-dead tissues treated with the negative control were also run as additional controls in the main test (for evaluation of the non-specific MTT reduction - NSMTT-). For each of these tissues, the NSMTT was calculated as follows:
NSMTT (%) = [(cODTIdead - mean cODNC_dead) / mean cODNC] x 100

With:
cODNC_dead = corrected OD of each freeze-dead tissue treated with the negative control,
cODTIdead = corrected OD of each freeze-dead tissue treated with the test item.

The mean NSMTT (%) was calculated and was subtracted to the mean relative viability of the test item-treated tissues to obtain the True MTT metabolic conversion, as follows:
True MTT metabolic conversion (%) = mean TI relative viability (%) - mean NSMTT (%)

The difference of viability between two tissue replicates was also calculated for each item.

Results and discussion

In vitro

Results

Irritation parameter:

other: % cell viability

Run / experiment:

mean

Value:

3

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

26%

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE CRITERIA
- the mean OD blank of each plate (i.e. extraction solvent) should be < 0.1,
- the mean cOD of the negative controls should be between 0.8 and 2.8,
- relative mean viability of the positive control should be < 50% of the relative mean viability of the negative control,
- the difference of viability between two tissue replicates should be < 20%.

EVALUATION OF THE COLOURATION OF TISSUES AT THE END OF THE MTT INCUBATION
The qualitative evaluation of the MTT staining was performed with the naked eye.
Viable test item-treated tissues appeared blue/white (i.e. white tissues with blue surrounds) which was considered to be indicative of semi-viable tissues.

EVALUATION OF MTT RESULTS
The relative mean viability of the tissues treated with the test item, corrected by the NSMTT, was 3% with a difference of 1% between duplicate tissues.
As the relative mean viability was < 60%, the acute eye irritation potential of the test item could not be predicted.

Applicant's summary and conclusion

Interpretation of results:

other: Between Category 1 (H318) and Category 2 (H319)

Conclusions:

Under the experimental conditions of this study, no prediction can be made on the acute eye irritation potential of the test item.
Accordingly, the classification of the test item should be Category 1/ Category 2 (GHS 2017) and Category 1 (H318)/Category 2 (H319) (Regulation (EC) No. 1272/2008).

Executive summary:

The purpose of this study was to predict the acute eye irritation potential of the test item by measurement of its cytotoxic effect on the EpiOcular^TMcornea epithelial model.

The design of this study was based on the OECD Test Guideline No. 492 and the study was conducted in compliance with Charles River Laboratories Evreux standard operating procedures and the principles of Good Laboratory Practice.

Methods

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Following the preliminary tests, the eye irritation potential of the test item was assessed in the main test. The test item and both negative and positive controls were applied topically on duplicate tissues and incubated at +37°C for 30 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes at room temperature to remove any remaining test item from the tissue, blotted on absorbent material, and then incubated for another 2 hours at, 5% CO₂in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (as reference viability).

Results

Preliminary tests

In the preliminary tests, the test item was found to have direct MTT reducing properties but no colouring potential.

Main test

All acceptance criteria were fulfilled. The study was therefore considered to be valid.

The calculated mean NSMTT was 0.35%.

The relative mean viability of the tissues treated with the test item (i.e.true MTT metabolic conversion, after correction by NSMTT) was 3% with a difference of 1% between duplicate tissues.

As the relative mean viability was < 60%, the acute eye irritation potential of the test item could not be predicted.

Conclusion

Under the experimental conditions of this study, no prediction can be made on the acute eye irritation potential of the test item.

Accordingly, the classification of the test item should be Category 1/ Category 2 (GHS 2017) and Category 1 (H318)/Category 2 (H319) (Regulation (EC) No. 1272/2008).