3-(p-methoxyphenyl)-2-methylpropionaldehyde

Endpoint summary

Description of key information

Skin corrosion: Not corrosive in absence of skin and eye irritation.

Skin irritation in vitro (OECD TG 439): not irritating
Eye irritation in vivo (similar to OECD TG 405): not irritating

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:

09 Nov 2016 to 14 Nov 2016

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)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

Test system:

human skin model

Source species:

human

Cell type:

other: EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model Kit

Justification for test system used:

Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model Kit
- Tissue batch number(s): 16-EKIN-045
- Supplier: SkinEthic Laboratories (Lyon, France)
- The EpiSkin™ tissues surface: 0.38 cm²

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: approximately 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
Each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- 2 mL assay medium containing 0.3 mg/mL MTT per well. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper.
- Incubation time: 3 hours at 37°C
- A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm

TEST FOR SUBSTANCE SPECIFIC INTERFERENCE IN MTT ASSAY
- Test for Direct MTT Reduction: 10 µL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue or purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.
- Assessment of Color Interference with the MTT endpoint: 10 µL of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL AND CONTROLS
10 µL

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min exposure with 10 µL undiluted substance

Value:

76.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also the substance is colorless.

Interpretation of results:

other: Not skin irritating

Remarks:

in accordance with EU CLP (1272/2008 and its updates)

Conclusions:

Under the conditions of this study the test item was not considered to be irritating to the skin, because the relative mean tissue viability was above 50% after 15 min exposure.

Executive summary:

The skin irritation potential of the test substance was tested in vitro using the EPISKIN™ after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The study procedures in the study were according to OECD TG 439 and GLP principles. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Triplicate tissues were treated with 10 µL undiluted test item for an exposure period of 15 minutes. Concurrent positive (5% aqueous SDS) and negative (DPBS) controls were included. After MTT-loading a biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. The optical density was measured at 562 nm. Data are presented as relative viability (%) (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 76.4%. Under the conditions of this study the test item was not considered to be irritating to the skin, because the relative mean tissue viability was above 50% after 15 min exposure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jul 1979 to 26 Jul1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Nicholas Helf
- Age at study initiation: At least 8 weeks
- Weight at study initiation: not specified
- Housing: 2/ cage in suspended wire mesh cages (30"x18"x 18")
- Diet: Fresh purina rabbit chow was freely available
- Water: Fresh water was freely available
- Acclimation period: At least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 21 °C
- Any extraneous material which might produce eye irritation was excluded from the area. The room, exclusively reserved for rabbits on acute tests, was kept clean in accordance with the standards of AAALAC.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
0.1 mL was placed in the conjunctival sac of the eye

Duration of treatment / exposure:

The test material was placed once in the conjunctival sac of one eye. The lids were held together briefly to insure adequate distribution of the test material.

Observation period (in vivo):

3 days

Number of animals or in vitro replicates:

6 animals (5 males, 1 female)

Details on study design:

OBSERVATIONS:
The ocular reactions of the cornea, iris and conjunctiva were graded at 1, 2, and 3 days after dosing.

SCORING SYSTEM: Draize scoring system. The scores were interpreted as defined in 16 CFR 1500.42.

Irritation parameter:

cornea opacity score

Basis:

animal: #1, #2, #3, #4, #5 and #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: #1, #2, #3, #4, #5 and #6

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 hours

Irritation parameter:

conjunctivae score

Basis:

animal: #2, #5

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

not fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal: #1, #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal: #3, #4, #5, #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Interpretation of results:

other: Not eye irritating

Remarks:

in accordance with EU CLP (1272/2008 and its updates)

Conclusions:

In an eye irritation study with rabbits, performed similar to OECD 405, no positive corneal , iridal or conjunctival scores were noted at any observation time. Based on the results of this study, the substance is not considered an eye irritant.

Executive summary:

A pre-guideline study, equivalent to OECD guideline 405, was performed to test eye irritation in six New Zealand White rabbits (at least 8 weeks old). 0.1 mL of undiluted test material was placed once in the conjunctival sac of one eye of each of the animals. The untreated eye severed as control. Observations were made after 24, 48 and 72 hours according to the Draize scoring system. No ocular reactions of the cornea and iris were observed. Ocular reactions of the conjunctiva (grade 1) were observed after 24 and 48 hours only, but were not assigned as positive test results. The results show that the substance is not irritating to the eye. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin corrosion: the substance is not corrosive because it is not skin and eye irritant. In addition, in the acute dermal toxicity study using 5000 mg/kg bw skin irritation was seen but not corrosion.

In vitro skin irritation (OECD TG 439)

The skin irritation potential of the test substance was tested in vitro using the EPISKIN™ after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The study procedures in the study were according to OECD TG 439 and GLP principles. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Triplicate tissues were treated with 10 µL undiluted test item for an exposure period of 15 minutes. Concurrent positive (5% aqueous SDS) and negative (DPBS) controls were included. After MTT-loading a biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. The optical density was measured at 562 nm. Data are presented as relative viability (%) (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 76.4%. Under the conditions of this study the test item was not considered to be irritating to the skin, because the relative mean tissue viability was above 50% after 15 min exposure.

In vivo eye irritation

A pre-guideline study, equivalent to OECD guideline 405, was performed to test eye irritation in six rabbits. 0.1 mL of undiluted test material was placed once in the conjunctival sac of one eye of each of the animals. The untreated eye severed as control. No ocular reactions of the cornea and iris were observed. Ocular reactions of the conjunctiva (grade 1) were observed after 24 and 48 hours only, but were not assigned as positive test results. The results show that the substance is not irritating to the eye. 

Respiratory irritation:

There are no occupational or consumer data indicating respiratory tract irritation. There are also no relevant experimental guidelines or results available that indicate respiratory irritation, therefore respiratory irritation is not anticipated. The ECHA guidance presents (R7a: 7.1.12.1) that respiratory irritation maybe be indicated when the substance is corrosive. The substance is not corrosive in the skin and is not an eye irritant (with irreversible damage) and therefore the substance is not a respiratory irritant.

Justification for classification or non-classification

Based on the available information classification and labelling for skin, eye and respiratory irritation is not warranted in accordance with EU CLP (EC 1272/2008 and its updates).