Tetrahydro-4-methyl-2H-pyran

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is classified as corrosive to the skin

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-14 to 2017-05-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

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 system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from a single donor

Source strain:

other: Normal human epidermal keratinocytes (NHEK), derived from neonatal-foreskin tissue

Details on animal used as source of test system:

TEST KIT
- Source: MatTex Corporation
- Type: Normal human epidermal keratinocytes (NHEK), derived from neonatal-foreskin tissue
- Lot no.: 25765

Cell culture media:
- Assay medium (MatTex Corporation)
- Lot no.: 041217CMHA
- Vehicle / postive controls: deionised water / 8N KOH (Wako Pure Chemical)

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

IN-LIFE DATES: 18 April 2017 to 19 April 2017

Vehicle:

unchanged (no vehicle)

Details on test system:

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 1 hour and observed visually after stirring.

Nylon mesh compatibility was also assessed, using the same methodology as outlined above. The nylon mesh was evaluated microscopically.

Procedure:
- Pre-incubation:
One 6-well plate was prepared for each 3 culture insert and the medium (0.9 mL) was added to each well of the plate and pre-incubated for 60 minutes in a CO2 incubator.

- Skin corrosion test:
3 and 60 minute exposures were conducted. 50 uL of the test article, vehicle (distilled water) and positive (8N KOH) controls were applied onto each tissue surface at 45 second intervals. Duplicate tissues were used for each dose level. After the exposure, a nylon mesh was placed on each tissue surface to spread the substances over the tissue surface. The 45 second interval allowed sufficient time for both application and washing procedures at the end of the exposure.

After the initial dosing of the 60 minute application, the plate was placed into the incubator for the remainder of the exposure period was reached.
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. The inserts were placed into a new 24 well plate filled with 300 uL/well of fresh medium.

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 tissues, MTT medium was removed and 2 mL of 2-propanol was added to the tissue inserts to allow formazan extraction from the tissues. Plates were placed into a plastic bag and extraction was performed at room temperature for 2 h or more using a plate shaker.

The 200 uL of 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. 2-propanol was used as a blank.

Cell viability was calculated as follows:
Cell viability (%) = (OD of each tissue / Mean OD of the negative control group) x 100

Tissue binding test was carried using the same procedure as described above, with the exception of medium without MTT was used. After the measuring of OD, the staining ratio was calculated by the following formula:
Staining ratio (%) = (OD of each tissue [without MTT] / Mean OD of the negative control group [without MTT]) x 100

Control samples:

yes, concurrent vehicle

Amount/concentration applied:

50 uL of test article

Duration of treatment / exposure:

single exposure / 3 and 60 minutes

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

2 replicates/dose

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

ca. 97.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

no indication of corrosivity

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

ca. 4.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

positive indication of corrosivity

Other effects / acceptance of results:

refer to "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

 

Skin corrosion:

The test article showed corrosivity effects during the prolonged exposure (60 minutes) following STEP 1 analysis. The mean relative tissue viability was >50% (97.4%) after the 3 minute exposure, but <15% (8.4%) after the 60 minute treatment (+ 42 hour post-incubation). In accordance with STEP 2 of the judgement criteria, as tissue viability was <25% following the 3 minute exposure, MTHP is deemed to be corrosive to skin, Category 1A.

 

The positive control viability was <15% (1.6%) after the 60 minute treatment, following a 42 hour post-incubation therefore confirming that corrosivity was detected with the test system.

 

The controls confirmed the validity of the study. The mean OD₅₅₀for the 3 minute and 60 minute negative control groups were 1.725 and 1.799, respectively.

 

Table CA 7.3.1/02-1: Summary of in vitro EPIDERM corrosivity result following application of MTHP

Parameter	Negative control	MTHP	Positive control
3 minute exposure
OD 570 nm	1.725	1.681	0.148
Mean relative tissue viability (%) ±CV	100 ±6.5	97.4 ±0.3	8.6 ±12.3
60 minute exposure
OD 570 nm	1.799	0.075	0.028
Mean relative tissue viability (%) ±CV	100 ±1.3	4.2 ±8.4	1.6 ±0.0

 

Deficiencies:

None.

 

Conclusion

Under the conditions of this study the MTHP showed corrosion effects. The mean relative tissue viability was>50% (97.4%) after the 3 minute exposure, but <15% (8.4%) after the 60 minute treatment (+42 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, MTHP is classified as Skin corrosion, Category 1A.

Interpretation of results:

Category 1A (corrosive) based on GHS criteria

Conclusions:

Under the conditions of this study the MTHP showed corrosion effects. The mean relative tissue viability was>50% (97.4%) after the 3 minute exposure, but <15% (8.4%) after the 60 minute treatment (+42 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, MTHP is classified as Skin corrosion, Category 1A.

Executive summary:

The potential of the test article to induce skin corrosion was analysed using the three-dimensional human skin model EPIDERM^TM comprising a reconstructed epidermis with a functional stratum corneum.

 

In the present study MTHP was applied topically to the EPIDERM^TM tissue for 3 and 60 minutes followed by a 42 hour post-incubation period and immediate determination of cytotoxic effects via the MTT reduction assay.

 

Corrosivity 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, 8N potassium hydroxide viability was <20% following a 60 minute exposure, thereby confirming that corrosivity could be detected in this test system.

 

The test article showed corrosivity effects during the prolonged exposure (60 minutes). The mean relative tissue viability was >50% (97.4%) after the 3 minute exposure, but <15% (8.4%) after the 60 minute treatment (+ 42 hour post-incubation). In accordance with STEP 2 of the judgement criteria, as tissue viability was <25% following the 3 minute exposure, MTHP is deemed to be corrosive to skin, Category 1A.  

Under the conditions of this study the MTHP showed corrosion effects. The mean relative tissue viability was>50% (97.4%) after the 3 minute exposure, but <15% (8.4%) after the 60 minute treatment (+42 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, MTHP is classified as Skin corrosion, Category 1A.