Tetrahydro-4-methyl-2H-pyran

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-12-07 to 2017-02-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

2015

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydro-4-methyl-2H-pyran

EC Number:

225-207-5

EC Name:

Tetrahydro-4-methyl-2H-pyran

Cas Number:

4717-96-8

Molecular formula:

C6H12O

IUPAC Name:

4-methyltetrahydro-2H-pyran

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 4-methyltetrahydropyran (MTHP)
- CAS no.: 4717-96-8
- EC-no.: not assigned
- Source and lot/batch No.of test material: Kuraray / MTHP-72715
- Expiration date of the lot/batch: not stated
- Molecular weight: 100.16 g/mol
- Purity: >99%

Test animals / tissue source

Species:

other: EpiOcular (reconstructed human cornea-like epithelium)

Strain:

other: supplied by MatTex Corporation

Details on test animals or tissues and environmental conditions:

TEST KIT
- Source: MatTex Corporation
- Type: Reconstructed human cornea-like epithelium
- Lot no.: 20973

Cell culture media:
- Assay medium (MatTex Corporation)
- Lot no.: 120516MWKC
- Vehicle / postive controls: deionised water / Methyl acetate (MatTex Corporation)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C, humidified incubator
- CO2 (%): 5%

IN-LIFE DATES: 12 December 2016 to 13 December 2016

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 uL
- Concentration (if solution): dosed as recieved (neat)

VEHICLE
- Amount(s) applied (volume or weight with unit): 50 uL
- Concentration (if solution): n/a
- Lot/batch no. (if required): municipal water purified using a water purifier to yield deionised water. Deionised water filter sterilised using a 0.22 um filter.
- Purity: sterile

Duration of treatment / exposure:

30 minutes / application to tissue surface

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

2 replicates/treatment group

Details on study design:

Pre-experimental checks:
To check the non-specific MTT-reducing capability of the test article 50 µL of the test article was mixed with 1 mL MTT (1 mg MTT/mL) medium and incubated for 3 hours and observed visually after stirring. For comparison, only 1 mg/mL MTT solution was treated in a similar manner

Procedure:
- Pre-incubation:
One 6-well plate was prepared for each treatment, 2 culture inserts and the medium (1.0 mL) was added to each well of the plate and pre-incubated for 60 minutes in a CO2 incubator. After 60 minutes of pre-incubation the plates were taken out of the CO2 incubator and culture inserts were transferred to the lower wells of the 6-well plates. The plates were incubated for 18-19 h in CO2 incubator.

- Exposure to the test material and rinsing:
After pre-incubation tissues were treated with each dose group in duplicate, starting with the negative control. The test article (50 µL) was added to the surface of the tissues. When the test materials were not spread over the entire tissue surface, the culture inserts were gently tapped to penetrate the test material into the entire tissue. The exposure time was 30 minutes in a CO2 incubator.

At the end of the exposure period tissues were washed with PBS to remove any residual test article. Excess PBS was removed by blotting bottom with blotting paper. After completion of rinsing, the culture inserts were promptly transferred to a 12 well plate containing medium and left to stand for 12 minutes. At the end of the 12 minute period, the inserts were transferred to a 6-well plate containing medium and incubated for 2 h in a CO2 incubator.

Cytotoxicity analysis (MTT):
Following the post-incubation period, the inserts were transferred in a prepared 24-well plate containing 300 µL pre-warmed MTT medium (1 mg/mL) and further incubated for 3 h, under the same conditions as previously stated.

After the 3 h MTT incubation period, the inserts were transferred to a 24-well plate containing isopropanol (2 mL/well) in order to extract the formazan. Extraction was carried out protected from light at room temperature for 2 hours.

The extract were transferred into a 96-well plate and the optical density was measured at 570 nm without reference wavelength in a plate spectrophotometer.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

ca. 4.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

refer to "Any other information on results incl. tables"

Any other information on results incl. tables

Formulation analysis:

None conducted.

 

Pre-experiment:

The mixture of test article and MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.

 

The mixture of the test article and aqua destain showed no colouring detectable by unaided eye-assessment.

 

Eye Irritation:

The test article showed irritant effects. The mean relative tissue viability was <60% (4.3%) after 30 minute treatment (+2 hour post-incubation). The positive control viability was <50%, therefore confirming that irritants were detected with the test system.

 

The controls confirmed the validity of the study. The mean OD₅₅₀of the vehicle control values was 1.279. The mean relative tissue viability of the positive control was <50% (38%). The difference in viability of each dose group was <20%.

 

Table CA 7.3.2/01-1:
Summary of in vitro EpiOcular result following application of MTHP

Parameter	Negative control	MTHP	Positive control
Mean OD 570 (difference)	1.279 (0.097)	0.055 (0.004)	0.481 (0.112)
Mean relative tissue viability (%) (difference)	100 (7.6)	4.3 (0.4)	37.6 (8.8)

 

Deficiencies:

None.

 

Conclusion

Under the conditions of this study MTHP showed irritant effects. The mean relative tissue viability was <60% (4.3%) after a 30 minute exposure (+ 2 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, MTHP requires classification and labelling. However, this test guideline cannot resolve between GHS Categories 1 and 2, typically therefore further classification on eye irritation/corrosion is required to decide on the final classification. However, MTHP is confirmed to cause skin corrosivity, therefore no further testing is required for ocular irritation/corrosion, with MTHP considered corrosive to the eye.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

MTHP is corrosive to skin, therefore no further testing is required for ocular irritation/corrosion, with MTHP considered corrosive to the eye

Conclusions:

Under the conditions of this study MTHP showed irritant effects. The mean relative tissue viability was <60% (4.3%) after a 30 minute exposure (+ 2 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, MTHP requires classification and labelling. However, this test guideline cannot resolve between GHS Categories 1 and 2, typically therefore further classification on eye irritation/corrosion is required to decide on the final classification. However, MTHP is confirmed to cause skin corrosivity, therefore no further testing is required for ocular irritation/corrosion, with MTHP considered corrosive to the eye.

Executive summary:

The potential of the test article to induce ocular irritation was analysed using the three-dimensional human eye EpiOcular model comprising a reconstructed human cornea-like epithelium.

 

In the present study MTHP (Tetrahydro-4 -methyl-2H-pyran) was applied topically to the EpiOcular tissue for 30 minutes followed by a 2 hour post-incubation period and immediate determination of cytotoxic effects via the MTT reduction assay.

 

Irritant potential of the test article was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with distilled water. The positive control, methyl acetate viability was <50%, thereby confirming that irritants could be detected in this test system.

 

The test item showed irritant effects. The mean relative tissue viability (% vehicle control) was <60% (21%) after 30 minute treatment and 2 hour post incubation.

 

Under the conditions of this study MTHP showed irritant effects. The mean relative tissue viability was <60% (4.3%) after a 30 minute exposure (+ 2 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, MTHP requires classification and labelling. However, this test guideline cannot resolve between GHS Categories 1 and 2, typically therefore further classification on eye irritation/corrosion is required to decide on the final classification. However, MTHP is confirmed to cause skin corrosivity, therefore no further testing is required for ocular irritation/corrosion, with MTHP considered corrosive to the eye.