2-isobutyl-4-methyltetrahydro-2H-pyran-4-yl acetate

Endpoint summary

Description of key information

Skin:

In an in vitro study, the reconstructed human epidermis test system (EpiDerm^TM, EPI-200) a tissue viability of 73.2 % was observed (BASF SE, 2017).

Eye:

In an in vitro eye irritation study (OECD 437) an In Vitro Irritancy score (IVIS) of 1.6 was determined (BASF SE, 2017).

In an in vitro Reconstructed Human Cornea like Epithelium (RhCE) eye irritation study a tissue viability of 49.8 % was observed (BASF SE, 2017).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch identification ZH 291 A1 Fr.2-4

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm TM, EPI-200, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue batch number(s): Lot Number 23362
- Date of initiation of testing: 2016-09-06

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (25 min, laminar flow hood) followed by 37 °C (35 min, incubator)
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: at least one washing step with sterile PBS
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES:
3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: freezing
- N. of replicates : 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if the mean relative tissue viability with a test material is less than or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL PBS, sterile

POSITIVE CONTROL
- Amount applied: 30 µL sodium dodecyl sulfate in water
- Concentration: 5 % (w/v)

Duration of treatment / exposure:

25 min (laminar flow hood) followed by 35 min (incubator)

Duration of post-treatment incubation (if applicable):

42 ± 4 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

73.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

 

		Tissue 1	Tissue 2	Tissue 3	Mean	SD	CV [%]
Test substance	Mean OD570	1.248	1.377	1.375	1.333
	Viability [% of NC]	68.5	75.6	75.5	73.2	4.1	5.6
NC	Mean OD570	1.973	1.838	1.654	1.821
	Viability [% of NC]	108.3	100.9	90.8	100.0	8.8	8.8
PC	Mean OD570	0.054	0.063	0.065	0.060
	Viability [% of NC]	3.0	3.4	3.5	3.3	0.3	9.2

 

The negative results derived with the Skin Irritation Test on pyranyl acetate pure alone were sufficient for a final assessment. Therefore further testing in the Skin Corrosion Test was waived.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ZH 291 A1 Fr.2-4

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source of collected eyes:
- Source: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Age at study initiation: > 12 months and < 60 months

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL

Duration of treatment / exposure:

10 min

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period the medium in both chambers was replaced with fresh pre-warmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 548 opacity units were discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
yes, 750 µLwater

POSITIVE CONTROL USED
yes, 750 µL ethanol (PC1), 750 µL dimethylformamide (PC2)

APPLICATION DOSE AND EXPOSURE TIME
750 µL, 10 min

POST-INCUBATION PERIOD:
no

REMOVAL OF TEST SUBSTANCE
Number of washing steps after exposure period: at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red).

POST-EXPOSURE INCUBATION:
2 h

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: The optical density (OD490) of passaged sodium fluorescein dye was determined with the aid of a SunriseTM Absorbance Reader.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Evaluation of the test results based on to the criteria given in OECD 437 (July 2013).

Irritation parameter:

in vitro irritation score

Value:

1.6

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Test substance identification	Mean Opacity Value	Mean Permeability Value	Mean In Vitro Irritacy Score
Test substance	1.5	0.003	1.6
NC	5.7	0.005	5.8
PC1 (Ethanol)	25.6	0.610	34.7
PC2 (DMF)	105.7	0.472	112.8

 

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ZH 291 A1 Fr.2-4

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

human

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

2

Details on study design:

- RhCE tissue construct used, including batch number:
EpiOcularTM model (OCL-200), Tissue Lot Number: 23734, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

- Doses of test chemical and control substances used
Test substance: 50 µL
Negative control (NC): 50 µL sterile, de-ionized water
Positive control (PC): 50 µL neat methyl acetate

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
Exposure: 30 min at 37 °C in the incubator
Post-exposure immersion: 12 min in prewaremed medium
Post-exposure incubation: 2 h at 37 °C in the incubator

- Number of tissue replicates used per test chemical and controls (positive control, negative control)
2

- Wavelength used for quantifying MTT formazan
570 nm

- Description of the method used to quantify MTT formazan: The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

Acceptance criteria:
Barrier function and Quality control (QC):
The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 (min) value following exposure to 100 µL of 0.3% Triton X-100 for each EpiOcular™ EIT (OCL-200) batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD Guideline.

Lower acceptance limit: ET50 = 12.2 min
Upper acceptance limit: ET50 = 37.5 min

NC: The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is > 0.8. The mean OD570 of the NC should not exceed 2.5.
PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable
Tissue variability: Two tissues were treated under the same conditions. A relative difference of both viabilities of < 20% is considered to be acceptable.

Irritation parameter:

other: cell viability (%)

Value:

49.8

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

 

		Tissue 1	Tissue 2	Mean	Inter-tissue variability [%]
Test substance	Mean OD570	0.861	0.866	0.864
	Viability [% of NC]	49.6	49.9	49.8	0.3
NC	Mean OD570	1.787	1.685	1.736
	Viability [% of NC]	103.0	96.0	100.0	5.9
PC*	Mean OD570	0.020	0.298	0.298
	Viability [% of NC]	1.2	17.2	17.2

 

*Due to mechanical damage of tissue 1 of the positive control (PC) during the washing procedure, only one tissue of the PC could be evaluated. Since the results of tissue 2 of the PC is well within the historical control data and all other acceptance criteria were met in the test, the outcome of the study is not expected to be influenced by this deviation.

 

 

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin:

The objective was to assess the skin irritation and corrosion potential of pyranyl acetate pure. Using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential of a substance. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT) according to OECD Guideline 439. However, in the current case for pyranyl acetate pure the results derived with SIT alone were sufficient for a final assessment. Therefore the SCT was waived.

The potential of pyranyl acetate pure to cause dermal irritation was assessed by a single topical application of 30 µL (irritation test) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues was compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. The following results were obtained in the EpiDerm™ skin irritation test:

After an exposure period of 1 hour with about 42 h post-incubation, the mean viability of the test-substance treated tissues was determined at 73.2% (values for single tissues: 68.5%, 75.6% and 75.5%). All acceptance criteria were met.

Based this and applying the evaluation criteria it was concluded, that pyranyl acetate pure does not show a skin irritation potential in the EpiDerm™in vitro skin irritation test. For this reason, corrosion to skin could be excluded and the respective test was waived.

 

Eye:

The objective of the present study was to determine a possible eye irritating potential of the test substance. A single in vitro assay may not always be sufficient, using the currently available methods, to cover the full range of eye irritating potential. Consequently, two in vitro assays were performed: The Bovine Corneal Opacity and Permeability Test (BCOP Test) according to OECD guideline 437 and the EpiOcularEye Irritation Test according to OECD guideline 492.

 

BCOP

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL test substance to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test substance preparation for an exposure period of 10 min and a post treatment incubation of 2 h. In addition to the test substance 750 µL of a negative control (NC; de-ionized water) and of two positive controls (PC1: ethanol, PC2: dimethylformamide) were applied to three corneas each. After the exposure period the corneas were washed at least 3 times with Eagle`s MEM. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. The following results were obtained in the BCOP Test:

 

	Mean Opacity Value	Mean Permeability Value	Mean In Vitro Irritancy Score
Test Substance	1.5	0.003	1.6
NC	5.7	0.005	5.8
PC1	25.7	0.610	34.7
PC2	105.7	0.472	112.8

 

 

EpiOcular

The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissue samples were incubated with the test substance for 0.5 hours followed by a 2 hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazoliumsalt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly.

TheEpiOcular™ eye irritation test showed the following results: The mean viability of the test-substance treated tissues was 49.8 %.

 

Summary of the individual test results of the in vitro eye irritation turnkey testing strategy:

 

Test Method	Test Result	Test Evaluation	Evaluation of the Test Strategy
BCOP Test	The mean IVIS of the test substance treated corneas was 1.6.	Not identified as corrosive or severe irritant.	Irritant
EpiOcularTM	The mean viability of the test substance treated tissues was 49.8 %.	Irritant

 

Based on the results for BCOP and EpiOcular Test and considering the evaluation criteria, the test substance showed an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

No skin irritating properties but eye irritating properties were identified. As a result the substance is classified as eye irritating (category 2, H319) under Regulation (EC) No. 1272/2008, as amended for the ninth time in Regulation (EC) No. 2016/1179.