p-methoxybenzyl acetate

Endpoint summary

Description of key information

Skin irritation: The test substance was determined to be non-irritant in an in vitro human skin model test with EpiDerm.

Eye irritation: The substance was considered to be non-irritating to rabbits' eyes.

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

Study period:

2015-10-27 to 2015-11-23

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:

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

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:

In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase onversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200- SIT Kit
- Tissue batch number: Lot No.: 23303
- Delivery date: 2015-11-17
- Date of initiation of testing: 2015-11-17 (pre-incubation)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 ± 1.5 °C, 25 min at room temperature
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: at least 15 times, volume not provided
- Observable damage in the tissue due to washing: no data provided
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm
- Filter: 570 ± 1 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive Contro: mean 4.77 %, range 4.00 % - 5.90 %

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean tissue viability is ≤ 50 %.
- The test substance is considered to be non-irritant to skin if the mean tissue viability is > 50 %

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 μL (47 μL/cm2)
- Concentration: unchanged

NEGATIVE CONTROL
- Amount applied: 30 μL DPBS

POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5 % SLS solution in deionised water

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

post-treatment: 42 hours and 38 minutes
MTT: 3 h treatment and 69 h extraction

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

103.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4.4 % tissue viability

Remarks on result:

no indication of irritation

Other effects:

The optical pre-experiment to investigate the test item’s colour change potential in water did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Dose Group	Exposure Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Rel. Absorbance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Mean rel. Absorbance [% of Negative Control]***
Negative control	1 h	1.402	1.549	1.502	1.484		5.1	100
Positive control	1 h	0.066	0.064	0.067	0.066		2.7	4.4
Test item	1 h	1.677	1.27	1.657	1.535		14.9	103.4

* Mean of three replicate wells after blank correction

** relative absorbance [rounded values]: (100x(absorbance tissue)/ mean absorbance negative control

*** relative absorbance [rounded values]: (100x(mean absorbance test item/positive control))/ mean absorbance negative control

 

The mean relative absorbance value of the test item, corresponding to the cell viability, was 103.4 % (threshold for irritancy:≤50 %), consequently the test item was not irritant to skin.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was determined to be non-irritant in the in vitro human skin model test with EpiDerm.

Executive summary:

The in vitro Human Skin Model Test with EpiDerm study according to OECD 439 was performed to assess the irritation potential of the test substance. Independent triplicate tissues of EpiDerm were exposed to either the test item, the negative control (DPBS) or the positive control (5 % SDS in water) for 1 hour. 30 μL of the test item were dispensed directly onto triplicate EpiDerm tissue surface, and spread to match the surface of the tissue. After exposure of the tissues to the test substance the mean tissue viability was 103.4 % after 1 h exposure. This value is above the threshold for irritation, which is defined to be < 50 %. Therefore, the test substance was considered to be not skin irritating. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test was not irritating to skin according to EU CLP and UN GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-04-04 to 2000-04-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

other: Little russian

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 1.9 - 3.0 kg bw
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL
- Concentration: undiluted

Duration of treatment / exposure:

single application

Observation period (in vivo):

1, 24, 48 and 72 h

Number of animals or in vitro replicates:

4 females

Details on study design:

SCORING SYSTEM: According to OECD guideline (draize scoring system)

TOOL USED TO ASSESS SCORE: hand held lamp fitted with white and UV-light; additionally fluorescein was used

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

The cornea score of eyes which were treated with fluorescein after the first 24 h reading was zero for all animals.

Interpretation of results:

GHS criteria not met

Conclusions:

The test material was considered to be not irritating to rabbits' eyes.

Executive summary:

The eye irritating potential ot the test material was investigated in a GLP-conform study according to OECD guideline 405. Four female albino rabbits were exposed to 0.1 mL of the test material in the left eye. Eye reactions were observed after 1 h, 24, 48 and 72 hours after expsoure. Slight signs of effects were observed on the treated eyes, but all effects were fully reversible within 72 hours. The mean scores for cornea opacity and iris score calculated for the time points 24, 48 and 72 hours were 0 for all animals. The conjunctiva score were calculated to be 0.33 for all animals. The chemosis score was calculated to be 0 for animal 1 and 0.33 for the three remaining animals. Based on these results, the material was not irritating to rabbits' eyes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation

Key study

An in vitro skin irritation test (OECD 439) was conducted to evaluate the skin irritation potential of the test substance. Undiluted test substance (30 µL) was applied to the human skin modell (EpiDerm) and incubated for 1 h. The viability of cells was evaluated with MTT test and measurement of optical density. As the mean viability was determined to more than 50 %, to be precise 103.4 %, the test substance is considered to be not skin irritating.

 

Supporting studies

Two studies in human volunteers were published. For details please refer to IUCLID section 7.10.5

Smith 2000: A volunteer study to evaluate skin irritation potential of the test substance was conducted with 10 healthy human volunteers (6 male, 4 female). A solution of 20 % test substance (1:4 in petrolatum), approximately 20 µg, as applied to the upper back using occlusive aluminum chambers secured with non-occlusive paper tape. Patches were applied for 24 h. Irritation was measured visually 50 min after patch removal and erythema and edema were scored. None of the 10 volunteers showed signs of skin irritation after 24 h treatment with the test substance. Therefore the test substance is considered to be not irritating to human skin.

Takenaka 1968: A patch test with 80 Japanese was conducted with the test substance. The application time was between 24 to 48 h and the concentration used was 0.03-0.5 % in base cream. Evaluation of the test side 30 min after exposure determination showed no skin irritating potential of the test substance.

 

Conclusion

The in vitro skin irritation test (OECD 439, key study) determined that the test substance had no skin irritating potential on the human skin modell. This result is supported by patch test studies with human volunteers, as no signs of irritation were detected on the skin of the volunteers.

 

Eye irritation

The eye irritating potential of the test material was investigated in a GLP-conform study according to OECD guideline 405. Four female albino rabbits were exposed to 0.1 mL of the test material in the left eye. Eye reactions were observed after 1 h, 24, 48 and 72 hours after exposure. Slight signs of effects were observed on the treated eyes, but all effects were fully reversible within 72 hours. The mean scores for cornea opacity and iris score calculated for the time points 24, 48 and 72 hours were 0 for all animals. The conjunctiva score were calculated to be 0.33 for all animals. The chemosis score was calculated to be 0 for animal 1 and 0.33 for the three remaining animals. Based on these results, the material was not irritating to rabbits' eyes.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin and eye irritation/corrosion, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the ninth time in Regulation (EU) No 2016/1179.