3-(p-cumenyl)propionaldehyde

Endpoint summary

Description of key information

Skin corrosion: The substance is not considered to be corrosive based on the absence of strong reactive groups (e.g. acids or bases), the absence of eye irritation and the absence of corrosive properties in the LLNA assay.

Skin irritation in vitro (OECD TG 439): irritating

Eye irritation in vitro (OECD TG 438): 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:

24 Oct 2016 to 25 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:

July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 23, 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

ENVIGO CRS GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf

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:

Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin models EpiSkin™ and EpiDerm and measurement of cell viability by dehydrogenase conversion 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: EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model Kit
- Supplier: SkinEthic Laboratories (69007 Lyon, France)
- The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
- EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels.
- Delivery date: 22 November 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37±1.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the inserts were immediately removed from the 12- well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- 2 mL assay medium containing 0.3 mg/mL MTT per well.
- Incubation time: 3hrs at 37±1.5°C
- Extraction of formazan: The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for nearly 3 hours while shaking at room temperature.
- Spectrophotometer: Microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1)
- Wavelength: 570 nm

TEST FOR DIRECT MTT REDUCTION AND COLOUR INTERFERENCE
- Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose the colourless test item (10 µL) was mixed with 90 µL of deionised water in a pre-experiment. The test item/water mixture was gently shaken for 15 minutes at room temperature.
- For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability 10 µL of the test item was added to 2 mL of MTT solution (0.3 mg/mL) and the mixture will be incubated in the dark at 37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution with 10 µL of DMEM was used as the control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µL

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42.25 hrs

Number of replicates:

Triplicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min exposure with 10 µL undiluted substance

Value:

23.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 (0.612-0.617) for the 15 minutes treatment interval thus showing the quality of the tissues.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.3% (< 40%) thus ensuring the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The rel. standard deviations between the % variability values of the test item, the positive and negative controls were below 13% (≤ 18%), thus ensuring the validity of the study.

Interpretation of results:

other: Skin irritant category 2

Remarks:

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

Conclusions:

Under the conditions of this study the test item was considered to be irritating to the skin, because the relative mean tissue viability was below 50% after 15 min exposure.

Executive summary:

The skin irritation potential of the test substance was tested in vitro using the EPISKIN™ model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42.25 hours. The study procedures 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% SLS solution in deionized water) and negative (deionized water) controls were included. After MTT-loading a biopsy of each epidermis was made and placed into micro tubes containing isopropanol containing 0.04 N HCl for extraction of formazan crystals out of the MTT-loaded tissues. The optical density was measured at 570 nm.Tests for direct MTT reduction and colour interference were negative.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 23.8%. Under the conditions of this study the test item was considered to be irritating to the skin, because the relative mean tissue viability was below 50% after 15 min exposure.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Aug 2016 to 21 Sep 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion B.V., Utrechtseweg 48, 3700 AV, Zeist

Species:

other: eyes of male or female chickens (ROSS, spring chickens)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Characteristics of donor animals: Approximately 7 weeks old, male or female chickens, body weight range approximately 1.5-2.5 kg, were used as eye donors.
- Storage, temperature and transport conditions of ocular tissue: Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- Time interval prior to initiating testing: Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus.
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 μL undiluted test substance

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

Negative control: 1
Positive control: 3
Test group: 3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min. The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 °C (water pump set at 36.4 °C). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

OBSERVATION PERIOD
240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline. After rinsing, each eye in the holder was returned to its chamber.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Slit-lamp microscope examination
- Damage to epithelium based on fluorescein retention: Slit-lamp microscope examination
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: set at 0.095 mm
- Others: After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

SCORING SYSTEM:
Defined scoring scales were used for each parameter to define the severity of effects into four categories (I-IV).
- Mean corneal swelling (%): According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean maximum opacity score: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean fluorescein retention score at 30 minutes post-treatment: According to OECD 438 guideline.

DECISION CRITERIA: According to OECD 438 guideline

Irritation parameter:

percent corneal swelling

Run / experiment:

slit-lamp examination

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

cornea opacity score

Run / experiment:

slit-lamp examination

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

fluorescein retention score

Run / experiment:

slit-lamp examination

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Other effects / acceptance of results:

Slit-lamp examination: The test substance caused corneal effects consisting of very slight corneal swelling (mean of 1%), very slight opacity (mean score of 0.5) and very slight fluorescein retention (mean score of 0.5).
The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.
The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight or severe erosion and slight or moderate vacuolation of the epithelium, the epithelium partly detached from the basement membrane (two corneas), and endothelial necrosis (two corneas).

Interpretation of results:

other: Not eye irritating

Remarks:

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

Conclusions:

Under the test conditions (OECD 438 and GLP) the test substance is not considered to be an eye irritant

Executive summary:

In accordance with OECD guideline 438 and GLP, the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused corneal effects consisting of very slight corneal swelling (mean of 1%), very slight opacity (mean score of 0.5) and very slight fluorescein retention (mean score of 0.5). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance revealed very slight erosion of the epithelium in one cornea. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed moderate or severe erosion and slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas). Based on these results, the test substance is not considered to be eye irritating.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin corrosion

The substance is not considered to be corrosive based on the absence of strong reactive groups (e.g. acids or bases), the absence of eye irritation and the absence of corrosive properties in the LLNA assay.

OECD TG 439 (skin irritation in vitro):

The skin irritation potential of the test substance was tested in vitro using the EPISKIN™ model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42.25 hours. The study procedures 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% SLS solution in deionized water) and negative (deionized water) controls were included. After MTT-loading a biopsy of each epidermis was made and placed into micro tubes containing isopropanol containing 0.04 N HCl for extraction of formazan crystals out of the MTT-loaded tissues. The optical density was measured at 570 nm.Tests for direct MTT reductionand colour interference were negative.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 23.8%. Under the conditions of this study the test item was considered to be irritating to the skin, because the relative mean tissue viability was below 50% after 15 min exposure.

OECD TG 438 (eye irritation in vitro):

In accordance with OECD guideline 438 and GLP, the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused corneal effects consisting of very slight corneal swelling (mean of 1%), very slight opacity (mean score of 0.5) and very slight fluorescein retention (mean score of 0.5). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance revealed very slight erosion of the epithelium in one cornea. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed moderate or severe erosion and slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas).Based on these results, the test substance is not considered to be eye irritating.

Justification for classification or non-classification

According to EU CLP (EC No. 1272/2008 and its amendments) the substance has to be classified as a skin irritant Category 2 but not for eye. The following phrase will be applied: H315, "Causes skin irritation".