cis-2-methyl-4-propyl-1,3-oxathiane

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24-09-2014 to 26-09-2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well reported study performed under GLP; using a commercially available human corneal epithelium model to manufacturer guideline with no significant deviations. Guideline currently under validation by the ECVAM.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation EpiOcular™ Eye Irritation Test (OCL-200-EIT)

Deviations:

yes

Remarks:

Tissue viability in meeting the acceptance criterion: the mean OD of three tissues is used at OD >= 1.0 instead of mean of two tissues according to guideline; no significant effect on the reliability of the study

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2012; signature: April 2013

Test material

Test material information

Constituent 1

Reference substance name:

cis-2-methyl-4-propyl-1,3-oxathiane

EC Number:

261-699-8

EC Name:

cis-2-methyl-4-propyl-1,3-oxathiane

Cas Number:

59323-76-1

Molecular formula:

C8H16OS

IUPAC Name:

2-methyl-4-propyl-1,3-oxathiane

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Physical state: liquid
- Storage condition of test material: In the refrigerator at 2 - 8 °C
- Other: colourless

Test animals / tissue source

Species:

other: human-derived epidermal keratinocyte tissues

Strain:

not specified

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Three tissues were used for the positive and negative control groups.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 uL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

30 minutes

Number of animals or in vitro replicates:

Three tissues were used for each treatment group (test item, negative control and positive control)

Details on study design:

- Details of the test procedure used: EpiOcular™ tissues were shipped at 2- 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt, EpiOcular™ tissues were kept in the refrigerator. After 24 hours and at least 1 hour prior to start of the assay, tissues were transferred to 6-well plates with 1 mL assay medium, pre-incubated, and transferred again to 6-well plates with assay medium. Prewarming: All incubations were done in the incubator at 37 ± 1.5 °C, 5 ± 0.5% CO2. Treatment: 37 ± 1.5 °C, 5 ± 0.5% CO2. MTT Assay: 37 ± 1.5 °C, 5 ± 0.5% CO2. After pre-incubation of EpiOcular™ tissues and after completion of the treatment with 20 uL PBS, the negative and positive control and the test item was added into the insert atop the concerning EpiOcular triplicate tissues. The plates were placed into the incubator for 30 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2. After the end of the treatment period the inserts were removed immediately from the cell culture plates. The tissues were rinsed with PBS for 15 times and submerged 3 times in PBS to remove any residual test material. The surface of the skin equivalents was carefully tried using cotton tips as well as by plotting the inserts with blotting paper. The inserts were then placed in 12-well plates supplemented with 5 mL medium per well. After 12 minutes incubation the inserts were transferred to 6-well plates containing 1 mL fresh medium per well. Following, the cell culture plates were placed in the incubator for about 120 minutes. Afterwards the cell viability was determined with the MTT assay.
- RhCE tissue construct used, including batch number: EpiOcular-Kit LotNo.: 19187, KitA
- Doses of test chemical and control substances used: 50 uL (neat test item)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
Exposure was for 30 minutes at room temperature.
Post exposure immersion: After the end of the treatment intervals the inserts were removed immediately from the cell culture plates. The tissues were gently rinsed with 15 times with each 1 mL PBS in order to completely remove any residual test material. The surface of the skin equivalents were carefully dried using cotton tips as well as by plotting the inserts with blotting paper. The inserts will then be placed in 12-well plates supplemented with 5 mL medium per well. After 12 ± 0.5 minutes incubation the inserts will be transferred to 6-well plates containing 1 mL fresh medium per well.
Post-exposure incubation: The cell culture plates will be placed in the incubator for 120 ± 15 minutes. Afterwards the cell viability will be determined with the MTT assay.
- Justification for the use of a different negative control than ultrapure H2O (if applicable): Not applicable.
- Justification for the use of a different positive control than neat methyl acetate (if applicable): Not applicable.
- Description of any modifications to the test procedure: Not applicable.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): Yes. Optical evaluation of the MTT -reducing capacity of the test item after 1 hour incubation with MIT-reagent did not show blue colour. The substance was considered to not directly reduce MTT.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): Triplicate.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm microplate reader.
- Description of the method used to quantify MTT formazan
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): Not applicable.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: Per SOP (and ECVAM validation).
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Yes (see full study report).
- Complete supporting information for the specific RhCE tissue construct used: Yes (see full study report).
- Reference to historical data of the RhCE tissue construct: Not available.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Not available.
- Positive and negative control means and acceptance ranges based on historical data: Yes (per reference literature).
- Acceptable variability between tissue replicates for positive and negative controls: Yes. Standard deviation was < 6.0%.
- Acceptable variability between tissue replicates for the test chemical: Yes. Standard deviation was < 2.0%.

SCORING SYSTEM:
A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of formazan formed, as monitored by the absorbance. The results for the time-dependant cytotoxicity curves can be presented as the arithmetic mean ± standard deviation. By setting the absorption of the negative control to 100% cell survival the relative absorption calculated for the test item and the positive control can also be described as tissue viability.

Tissue Viability = 100 x (absorption-testitem/positive control –Absorption-blank) / (Absoption-neg.control – Absoption-blank)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: TEST ITEM: Mean Relative Absorbance (% of negative control)

Remarks:

30 minutes (exposure)

Run / experiment:

mean

Value:

45.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

S.D. 10.5% (n=3)

Results 2

Irritation parameter:

other: POSITIVE CONTROL: Mean Relative Absorbance (% of negative control)

Remarks:

30 minutes (exposure)

Run / experiment:

mean

Value:

34.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

S.D. = 0.9 % (n=3)

Results 3

Irritation parameter:

other: NEGATIVE CONTROL: Mean Relative Absorbance (% of negative control)

Remarks:

30 minutes (exposure)

Run / experiment:

mean

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

S.D. = 5% (n=3)

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Concurrent positive control was within the expected range.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. Standard deviation was < 6.0%.
- Acceptance criteria met for positive control: Yes. Standard deviation was < 6.0%.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

Any other information on results incl. tables

Table 1 Results after treatment

Group	Mean Absorbance* Tissue 1, 2, 3 Minus Mean Blank	Mean Absorbance of 3 tissues	Relative Absorbance (%) Tissue	SD (%)	Mean Relative Absorbance (% of negative control)
Negative control	1.540	1.460	102.7	5.9	100
Negative control	1.559		104.0
Negative control	1.401		93.2
Positive control	0.546	0.498	34.6	2.2	34.1
Positive control	0.501		31.6
Positive control	0.501		35.9
Test item	0.729	0.668	47.2	1.3	45.8
Test item	0.702		45.3
Test item	0.692		44.7

* mean of three replicate wells after blank correction

** relative absorbance (rounded) : 100 x (absorbance-testitem/positivecontrol) / (absorbance-neg.control)

 

Optical evaluation of the MTT -reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. The test item was considered to not directly reduce MTT.

 

The relative absorbance values of the test item, corresponding to the cell viability, did not decrease below 60% compared with the result of the negative control.

 

The acceptability criteria) of the assay were met.

The corrected mean OD values of the three tissues exposed to the negative control were>= 1.0 (1.540, 1.559, 1.401)

The mean tissue viability of the positive control is 34.1% of the negative control (<= 60%)

The standard deviation of the three identically treated skin equivalents of each dose group was <= 20% (below 6%)

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

Under the conditions of this study the test substance is an eye irritant.

Executive summary:

The study was performed to assess the irritancy potential of the test item to the eye means of the Human Cornea Model Test using the EpiOcular™ model. The study was performed to GLP. The method was according to manufacturer guideline. Tissues of the human cornea model EpiOcular™ were pre-treated for 30 minutes with PBS. The tissues were exposed to each 50 uL of the test item and of the positive control: methyl acetate (neat) and the negative control (deionised water) for 30 minutes in triplicate. Afterwards the cell viability was determined using the MTT assay. After treatment with the negative control, the mean OD of the three tissues was >= 1.0. Compared with the value of the negative control, treatment with the positive control induced a sufficient decrease in the relative absorbance, and the mean tissue viability was <= 60% of the negative control. The relative standard deviation between each the three identically treated equivalents of each dose group was <= 20%. Consequently, the test met the acceptance criteria. Irritating effects were observed following incubation with test item with a mean tissue viability of 45.8%. The relative absorbance values of the test item, corresponding to the cell viability, decreased below 60% compared with the result of the negative control; consequently, the test item is considered to possess an eye irritating potential.