Bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-03 to 2012-11-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate

EC Number:

261-879-6

EC Name:

Bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate

Cas Number:

59719-67-4

Molecular formula:

C24H46N4O6

IUPAC Name:

bis[2-(2-isopropyl-1,3-oxazolidin-3-yl)ethyl] hexane-1,6-diylbiscarbamate

In vitro test system

Test system:

human skin model

Source species:

other: reconstructed human epidermis

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiSkinTM-SM model has been validated for irritation testing in an international trial and is considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTMSM, EPISKIN SNC Lyon, France
- Tissue batch number: 12-EKIN-038
- Expiry date: 22 October 2012
- Date of initiation of testing: 17 October 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time, the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging the epidermis).
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3 h
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irrtating to skin if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: The test item is a viscous liquid therefore exact weighting of treatment volume was not performed. While avoiding infinite dose a sufficient amount to cover evenly all the epidermal surface (approx. 20 μL) was spread gently with a flat curved spatula onto the skin.

NEGATIVE CONTROL
- Amount applied: 20 µL
- Concentration: 1 x

POSITIVE CONTROL
- Amount applied: 20 µL
- Concentration: 5 %

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

70

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be non-irritant to skin.

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method TOXI-COOP ZRT. demonstrated the technical proficiency, using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The mean OD value of the three negative control tissues was 0.648.
- Acceptance criteria met for positive control: yes. The mean OD value obtained for the positive control was 0.119 and this result corresponds to 18 % viability when compared to the results obtained from the negative controls.
- Acceptance criteria met for variability between replicate measurements: yes. Each calculated standard deviation value (SD) for the % viability was below 18.
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

VALIDITY OF THE TEST:
The mean OD value of the three negative control tissues was 0.648. The mean OD value obtained for the positive control was 0.119 and this result corresponds to 18 % viability when compared to the results obtained from the negative controls. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Indicator for potential false viability

Possible direct MTT reduction with test substance:
No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Colouring potential of test substances:
The test item showed no ability to become coloured in contact with water therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, Incozol 4 is considered as non-irritant to skin and is therefore not classified.

Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item Incozol 4 on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan crystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control. The test item is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal to (≤) 50% when compared to the viability values obtained from the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item Incozol 4 did not show significantly reduced cell viability in comparison to the negative control. All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, Incozol 4 is considered as non-irritant to skin and is therefore not classified.