2,2-dimethylpropanethioamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May -July 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

The purpose of this study was to determine the eye irritation potential of the test item using the SkinEthic Reconstructed Human Corneal model (RHC, SkinEthic Laboratories, Nice, France) following treatment periods of 10 and 60 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The experimental design of the study consists of a test for Direct Reduction of MTT by the test item, followed by the main test.
For the main test, triplicate SkinEthic tissues were treated with the test item and exposed for 10 minutes and 60 minutes. The tissues were incubated at 37°C in a humidified atmosphere of 5% CO2 in air for the appropriate exposure period.
Duplicate tissues served as the negative control or positive control for each exposure period.
At the end of the 10-Minute exposure period each SkinEthic tissue was rinsed using Dulbecco’s Phosphate Buffered Saline (DPBS) and placed into a ‘holding plate’ until all the tissues had been rinsed. The rinsed tissues (two per group) were then transferred to an MTT ‘loading plate’, and incubated at 37°C for 3 hours in a humidified atmosphere of 5% CO2 in air. The remaining test item treated tissue was retained for possible histology. Following MTT loading, each SkinEthic tissue was blotted dry and placed into an MTT ‘extraction plate’ in order to extract all of the reduced MTT from the tissues. The same rinsing, retention, loading and extraction procedures were repeated for the 60-Minute tissues once the 60-Minute exposure period was complete.
At the end of the extraction period, the extracted MTT solution was mixed for each SkinEthic tissue and 3 x 200 μL samples, representing each tissue, were transferred to the appropriate wells of a 96 well plate. The optical density at 540nm (OD540) of each well was measured. Data are presented in the form of percentage viability (MTT conversion relative to negative controls) for each of the two exposure periods.
The results were used to make a prediction of the eye irritation potential of the test item.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2-dimethylpropanethioamide

EC Number:

677-829-5

Cas Number:

630-22-8

Molecular formula:

C5H11NS

IUPAC Name:

2,2-dimethylpropanethioamide

Test material form:

solid: crystalline

Details on test material:

Purity or Composition 100.0 %

Test animals / tissue source

Species:

human

Strain:

other: SkinEthic Reconstructed Human Corneal model

Details on test animals or tissues and environmental conditions:

The SkinEthic RHC model consists of transformed human keratinocytes of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg

Duration of treatment / exposure:

10 and 60min

Number of animals or in vitro replicates:

3

Details on study design:

Triplicate tissues were treated with the test item for exposure periods of 10 minutes and 60 minutes. 30 mg of the test item was topically applied to the corresponding tissues. The tissues were dosed at regular timed intervals, to allow for the period taken to rinse each insert following exposure, and to ensure each tissue received an equal exposure time. Duplicate tissues were treated with 30 μL of solution A to serve as negative controls for the 10 and 60-Minute exposure periods. Duplicate tissues were treated with 30 μL of SDS 0.5% w/v to serve as positive controls for the 10 and 60-Minute exposure periods. In order to eliminate the possibility of volatile test items affecting the viability of other treated tissues, the plates were sealed with a gas permeable sealing membrane. The plates were incubated at 37°C, 5% CO2 in air for the respective exposure times.
At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing DPBS. Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated ‘holding plate’ containing 300 μL of maintenance medium (at room temperature) until all the tissues were rinsed (including the negative and positive control treated tissues). Following rinsing the tissues (two per group) were transferred to a pre-labelled 24-well plate designated ‘MTT Loading plate’ containing 300 μL of a 0.5 mg/mL MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for three hours at 37°C, 5% CO2 in air.
At the end of the incubation period the inserts were blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labelled 24-well plate designated ‘MTT extraction plate’ containing 0.75 mL of isopropanol in each of a sufficient number of wells. An extra 0.75 mL of isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminium foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Item - Relative Mean Viability %

Value:

94.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative Control Item - Relative Mean Viability %

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive Control Item - Relative Mean Viability %

Value:

69.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Applicant's summary and conclusion

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Conclusions:

The result of this study was combined with the result of a Rabbit Enucleated Eye Test, Harlan Laboratories Ltd Project number 41101184(2). The combined classification was Mild-Moderate Irritant, EU Risk Phrase R36 “irritating to eyes”, CLP Category 2, GHS: Category 2A.