4-(5-Propyl-tetrahydropyran-2-yl)-phenol

Endpoint summary

Description of key information

Eye irritation was studied in vitro in GLP complinet assays as listed below:

OECD 492: positive (Cat 1 or 2)

OECD 437: corrosive

Skin irritation was studied in vitro in GLP complinet assays as listed below:

OECD 439: positive (Cat 1 or 2)

OECD 431: negative

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 439.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECV AM in 2008.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number: 21-RHE-104
- Expiry date: June 28, 2021
- Date of initiation of testing: June 23, 2021

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 of washing steps: 25 mL DPBS
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800, BioTek (Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 1.3 (OD>0.7)
- Barrier function: 7.0 h (4.0h - Morphology: Multi-layered, highly differentiated epidermis consisting of basal, spinous and granular layers, and a multi-layered Stratum corneum.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- N. of replicates : 3
- Method of calculation used: It is necessary to evaluate the OD due to non-specific reduction and to subtract it before calculation of the cell viability.
Non specific MTT reduction = [OD(KT) - OD(KU) / OD (neg. control)] * 100
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3
PREDICTION MODEL
- The test substance is considered as non-irritant to skin (UN GHS No Category) if the tissue viability after exposure and post-treatment incubation is > 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg ± 2 mg per tissue
NEGATIVE CONTROL
- Amount applied: 16 μL ± 0.5 μL per tissue
POSITIVE CONTROL
- Amount applied 16 μL ± 0.5 μL per tissue

Duration of treatment / exposure:

42 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

2.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, but a grey to black discoloration was observed. It can therefore be assumed that the test item is a direct MTT reducer, but the test item has no colorant properties.
It was not necessary to perform a test on killed controls because any MTT reduction would not change the classification since viability is already below the cut-off of 50 %.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Interpretation of results:

other: classification and labeling required according to UN GHS (Category 2 or 1)

Conclusions:

Under the conditions of the present study, the test item is identified as requiring classification and labeling according to UN GHS (Category 2 or 1).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 431.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

reconstituted human epidermis (RHE)-model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The SkinEthic RHE-model RHE/S/17 was obtained from Episkin/SkinEthic Laboratories, Lyon, France.

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECV AM in 2008.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number: 21-RHE-138
- Expiry date: September 6, 2021
- Date of initiation of testing: September 1, 2021

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 of washing steps: 20 mL DPBS
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800, BioTek (Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 1.3 (OD>0.7)
- Barrier function: > 10.0 h (4.0h For the barrier function, the ET50 value is above 10.0 and thereby above the accepted range. However, though this acceptance criterion was not fulfilled, the quality of the membrane is considered sufficient based on the experience of the supplier.
- Morphology: Multi-layered, highly differentiated epidermis consisting of basal, spinous and granularlayers, and a multi-layered Stratum corneum.

NUMBER OF TISSUES:
The test item as well as the negative control were tested with two replicate tissues per time point (3 min and 1 hour exposure). The positive control was tested with two replicate tissues only for 1 hour. Additionally, the test item and the negative control were tested with two killed tissues per time point (3 min and 1 hour exposure), each.
Therefore, a total number of 18 tissues was used in this study.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

Solid test item: 20 ± 3 mg per tissue
Positive control: 40 ± 3 µL per tissue
Negative control: 40 ± 3 μL per tissue

Duration of treatment / exposure:

At room temperature for 3 minutes and 1 hour.

Duration of post-treatment incubation (if applicable):

MTT Assay 42 h

Number of replicates:

2 replicate tissues per time point (3 min and 1 hour exposure)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean, 1 h

Value:

100.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean, 3 min

Value:

108.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Other effects / acceptance of results:

OTHER EFFECTS:
The pre-test for direct MTT-reducing capacity of the test item did result in blue color, i.e. the test item is a direct MTT reducer, but the test item has no colorant properties.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 437.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

Fresh isolated bovine eyes of cattle were used in this study.
Slaughterhouse: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
Age of cattle: 14 - 23 months
Corneal diameter: 25 - 28 mm

Vehicle:

other: 0.9 % sodium chloride solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL of the suspended test item (i.e. 150 mg/750 µL), positive or negative control were applied on the corneas.

Number of animals or in vitro replicates:

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride Solution
POSITIVE CONTROL USED: 20 % imidazole in 0.9 % sodium chloride solution
APPLICATION DOSE: 750 μL
TREATMENT METHOD: closed chamber
POST-INCUBATION PERIOD: yes, 240 min
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity value of each individual cornea was corrected for background opacity by subtracting the initial baseline opacity reading from the post treatment opacity reading. In addition, the opacity values of both the treatment and positive control groups were corrected for the mean negative control opacity values.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA: The decision criteria as indicated in the OECD TG 437 were used.

IVIS
<3 No Category
>3<55 No prediction can be made
>55 Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

65.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (IVIS = 1.0)
- Acceptance criteria met for positive control: yes (IVIS = 96.1)

The IVIS obtained after treatment with the test substance was 65.3 and, thus, higher than 55, i.e. according to OECD 437 the test item is inducing serious eye damage (UN GHS: Category 1). 

No observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Under the conditions of the present study, the test item did show an eye hazard potential. The test item is inducing serious eye damage (UN GHS: Category 1).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 492.

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)

Species:

human

Details on test animals or tissues and environmental conditions:

TEST SYSTEM:
Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 34914
Keratinocyte strain: 4 F 118 8
Supplier: MatTek In Vitro Life Science Laboratories

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Solid test item: 50 mg per tissue

Negative control: 50 μL per tissue

Positive control: 50 μL per tissue

Duration of post- treatment incubation (in vitro):

6 h

Details on study design:

NUMBER OF REPLICATES: 2

NEGATIVE CONTROL USED: Deionised water

POSITIVE CONTROL USED: Methyl acetate

APPLICATION DOSE: 50 mg

POST-INCUBATION PERIOD: yes, 6 h
REMOVAL OF TEST SUBSTANCE
- At the end of the 6 hours treatment time, the positive control, negative control and the test item
were removed by extensively rinsing the tissues with pre-warmed (room temperature) DPBS.
Three clean beakers, containing a minimum of 100 mL each of DPBS were used per group.

EVALUATION OF RESULTS:
The calculation was performed with the MS-Excel-Sheet "lnVitroEyelrritation_ VOl ". The mean
OD (optical density) of the two negative control tissues as calculated. This value corresponds to
100% tissue viability in the current test. For each individual tissue treated with the test item or
the positive control the individual relative tissue viability was calculated according to the
following formula:
Viability (%) = [test item OD / mean negative control OD] * 100

Prediction Model according to OECD 492:
The test item is identified as not requiring classification and labeling according to UN GHS (No
Category) if the mean percent tissue viability is more than 60%. In this case no further testing in
other test methods is required.
If the mean percent tissue viability is less than or equal 60%, no prediction can be made. In this
case, further testing with other test methods will be required because RhCE test methods show a
certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.
Mean tissue viability > 60 % : No Category
Mean tissue viability <= 60 %

Irritation parameter:

other: Viability

Run / experiment:

Mean

Value:

6.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, but a
grey to black discoloration was observed. lt can therefore be assumed that the test item is a direct
MTT reducer, but the test item has no colorant properties.

ACCEPTABILITY OF THE TEST:

The study met all acceptable criteria.

Negative control OD:
Acceptable Criterion: > 0.8 and < 2.5
Result: 1.550 and 1.499

Mean viability positive control:
Acceptable Criterion: < 50 %
Result: 29.9 %

Difference of viability between the two relating tissues of a single chemical:
Acceptable Criterion: < 20 %
Result: 3.3 % (negative control), 4.3 % (positive control), 0.7 % (test item)

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, no prediction can be made regarding the eye irritating
potential of the test item.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available experimental information, the substance needs to be classified as skin irritant (Cat 2) and for irreversible effects to the eye (Cat 1) considering the criteria outlined in the EU Regulation (EC) No 1272/2008 or any updates on Classification,Labelling and Packaging of Substances and Mixtures.