4-trans-propenylveratrole

Endpoint summary

Description of key information

Skin irritation/corrosion: not irritating (OECD 439, GLP, K, rel. 1).

Eye irritation: not irritating (OECD 405, GLP, rel.1).

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:

From January 27 to May 18, 2015

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on July 01-03, 2014/ signed on September 15, 2014)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Following the REACH bottom-up strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 15-EKIN-019
- Production date: not reported
- Shipping date: 12 May 2015
- Delivery date: 12 May 2015
- Expiry date: 18 May 2015
- Date of initiation of testing: 12 May 2015

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Number of steps not reported. Each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS and then incubated in 2mL maintenance medium for 42 h at 37 °C, 5 % CO2 in air.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: BMG Fluostar Optima – plate reader
- Wavelength: 540 nm
- Filter: not reported
- Filter bandwidth: not reported
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control OD values: mean 0.811 ± 0.082 (mean historical OD of the negative control was 0.788 ± 0.082)
- Barrier function: IC50 ≥ 1.5 mg/ml (threshold value)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: absence of bacteria, fungus and mycoplasma
- Reproducibility: For the previous 57 experiments conducted between October 2008 and December 2014 using this test method, the mean OD of the positive control was 0.168 ± 0.076 and the mean percentage viability was 21.5 ± 9.6 (The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤18%). In this same period the mean OD of the negative control was 0.788 ± 0.082 (The assay establishes the acceptance criterion for an acceptable test if the mean OD 562 for the negative control treated tissues was ≥0.6 and ≤1.5).

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if relative mean tissue viability is ≤ 50% after 15 minutes of exposure.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is > 50% after 15 minutes of exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 μL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution

Duration of treatment / exposure:

The EpiSkin™ human epidermis skin constructs were treated with the undiluted test item for an exposure period of 15 minutes.

Duration of post-treatment incubation (if applicable):

At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 42 h.

Number of replicates:

Triplicate tissues for test item, negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure period and 42 h post-exposure incubation period

Value:

97.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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 (97.3 ± 10.7%)

Possible reduction of MTT by test substance: There was no change in the test substance/MTT solution or the water control/MTT solution after three hours incubation in the dark at 37±2 °C in a humidified atmosphere of 5% CO₂ in air. The test substance had not interacted with the MTT.

Check for colouring potential of test substance: The test substance/water solution and water control were colourless after the 15 minute shaking period. The test substance had not shown any potential for colouring water.

Table 7.3.1/1: EpiSkin^™results

Sample	Tissue viability as percentage of mean OD negative control				Prediction MTT endpoint
	Replicate Tissues			Mean ± SD
	a	b	c
Negative Control	92.3	95.2	112.6	100.0 ± 11.0	Not applicable
Positive Control	12.8	10.0	8.6	10.5 ± 2.1	Irritant
Test item	88.8	109.4	93.7	97.3 ± 10.7	Non-irritant

 

Assay validity

Negative control: The mean absorbance of the triplicate negative control values was 0.811 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 11.0 which was below the maximum value of 18.

Positive control: The percentage mean viability of the positive control was 10.5 ± 2.1 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18%.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, test item is not classified as a skin irritant according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, the undiluted test substance was applied to EpiSkin TM human epidermis skin constructs (triplicate tissues). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item. The constructs were treated with the neat test substance for 15 minutes. After rinsing of the test substance the constructs were incubated for 42 hours. The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5‑diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances.

This assay was valid with negative and positive controls showing results within the acceptable range.

It was concluded that the test substance with a mean tissue viability of 97.3 ± 10.7%, was predicted as non-irritant to the skin.

Under the test conditions, test item is not classified as a skin irritant according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

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:

From October 16 to 20, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

(inspected on June 14, 1999 / signed on December 01, 1999)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River, Deutschland GmbH, Stolzenseeweg 32-36, 88353 Kißlegg
- Weight at study initiation: 2.5-2.7 kg
- Housing: Animals were housed individually in PPO cages (floor area: 2576 cm2) with perforated floor.
- Diet: Pelleted complete rabbit diet (‘Altromin 2123’ from Altromin, D-32791 Lage, Lippe), ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 55 ± 15 %
- Air changes: 10 times / h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: October 16, 2000 To: October 20, 2000

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL

Duration of treatment / exposure:

24 h

Observation period (in vivo):

1, 24, 48 and 72 h after instillation of test material

Number of animals or in vitro replicates:

4 females

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Fluorescein was instilled into eyes of test animals after 24 h reading. Then eyes were rinsed with 20 mL of 0.9 % sodium chloride solution.
- Time after start of exposure: 24 h

SCORING SYSTEM: Draize scale as described in the OECD guideline No. 405.

TOOL USED TO ASSESS SCORE: Eyes (before and after instillation of fluorescein) were examined using hand held inspection lamp fitted with white and UV-light and magnifying glass with 2 x magnification.

Irritation parameter:

cornea opacity score

Basis:

animal: #1, #2, #3 & #4

Time point:

other: Mean 24, 48 and 72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: #1, #2, #3 & #4

Time point:

other: Mean 24, 48 and 72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal: #1 & #4

Time point:

other: Mean 24, 48 and 72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

other: Mean 24, 48 and 72 h

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

other: Mean 24, 48 and 72 h

Score:

0.67

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal: #1, #2, #3 & #4

Time point:

other: Mean 24, 48 and 72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

- 1 h after test material instillation, two animals showed conjunctival vessels definitely injected and a swelling above normal. A diffuse, crimson red conjunctiva with individual vessels not easily discernible as well as a swelling above normal was observed in one animal. One animal showed an iris with markedly deepened folds, congestion swelling, moderate circumcorneal injection and existence of reaction to light, a diffuse, crimson red conjunctiva with individual vessels not easily discernible as well as a swelling above normal was observed.
- 24 h after test material instillation, three animals showed some conjunctival vessels definitely injected. One animal showed no signs of eye irritation.
- 48 h after test material instillation, one animal showed some conjunctival vessels definitely injected. No eye irritation was observed in other animals.
- None of the four animals showed any sign of eye irritation at 72 h.

Other effects:

None

Table 7.3.2/1: Eye irritation response data for each animal at each observation time

Score at time point	Cornea		Iris (/2)	Conjunctivae
	Opacity (/4)	Area (/4)		Redness (/3)	Chemosis (/4)	Discharge (/3)
1 h	0/0/0/0	0/0/0/0	0/0/0/1	1/1/2/2	1/1/1/1	0/0/0/0
24 h	0/0/0/0	0/0/0/0	0/0/0/0	1/0/1/1	0/0/0/0	0/0/0/0
48 h	0/0/0/0	0/0/0/0	0/0/0/0	0/0/1/0	0/0/0/0	0/0/0/0
72 h	0/0/0/0	0/0/0/0	0/0/0/0	0/0/0/0	0/0/0/0	0/0/0/0
Average 24, 48 and 72 h	0/0/0/0	0/0/0/0	0/0/0/0	0.33/0/0.67/0.33	0/0/0/0	0/0/0/0
Reversibility	-	-	Completely reversible	Completely reversible	Completely reversible	-
Average time (unit) for reversion	-	-	24 h	72 h	24 h	-

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, the test material is not classified as irritating to the eyes according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In an eye irritation study performed according to the OECD Guideline No. 405, and in compliance with GLP, 0.1 mL of undiluted test material was instilled into one eye of 4 female New Zealand White rabbits. The other eye remained untreated and served as control. The upper and lower eyelids were held together for about one second immediately after instillation. The eyes were examined and the changes were observed at 1, 24, 48 and 72 h after treatment and graded according to the Draize method.

1 h after test material instillation, two animals showed conjunctival vessels definitely injected and a swelling above normal. A diffuse, crimson red conjunctiva with individual vessels not easily discernible as well as a swelling above normal was observed in one animal. One animal showed an iris with markedly deepened folds, congestion swelling, moderate circumcorneal injection and existence of reaction to light, a diffuse, crimson red conjunctiva with individual vessels not easily discernible as well as a swelling above normal was observed. 24 h after test material instillation, three animals were observed some conjunctival vessels definitely injected. 48 h after test material instillation, one animal showed some conjunctival vessels definitely injected. None of the four animals showed any sign of eye irritation at 72 h.

The calculated mean score for each animal within 3 scoring times (24, 48 and 72 h) were 0.33/0.0/0.67/0.33 for redness, 0/0/0/0 for chemosis, 0/0/0/0 for iris lesions and 0/0/0/0 for corneal opacity. The effects observed were all reversible within 72 hours.

Under the test conditions, the test material is not classified as irritating to the eyes according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for eye irritation endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Since no key study was identified on the registered substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (July 2015), was used to evaluate the skin corrosion/irritation potential of the registered substance:

	Element	Information	Conclusion	Comments
Existing data on physico - chemical properties	1a	Is the substance spontaneously flammable in contact with air (pyrophoric) or water at room temperature?	NO
	1b	Is the substance an organic hydroperoxide or an organic peroxide?	NO
	1c	Is the pH of the substance ≤ 2.0 or ≥ 11.5?	NO
	1d	Are there other physical or chemical properties that indicate that the substance is corrosive/irritant?	NO
Existing human data	2	Are there adequate existing human data which provide evidence that the substance is a corrosive or irritant?	NO	Slight irritation was sometimes observed in existing human patch tests. However, the substance was not applied undiluted, and therefore these results were not considered adequate
Existing animal data from corrosion/irritation studies	3	Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?	NO
Existing data from general toxicity studies via the dermal route and from sensitisation studies	4a	Is the substance classified as fatal in contact with skin (LD50 ≤ 50 mg/kg bw, CLP hazard statement H310)	NO	Dermal LD50 > 5000 mg/kg bw
	4b	Has the substance proven to be a corrosive, irritant or non-irritant in a suitable acute dermal toxicity test?	NO	Dermal irritation was reported in the acute dermal toxicity test, but scores were not reported, therefore a conclusion was not possible
	4c	Has the substance proven to be a corrosive or an irritant in sensitisation studies or after repeated exposure?	NO	LLNA available: tested only up to 50%. No sign of irritation or corrosion leading ot C&L. NB: In general, irritation data from the Local Lymph Node Assay are not usable. The test substance is applied to the dorsum of the ear by open topical application
Existing/new (Q)SAR data and read -across	5a	Are there structurally related substances (suitable “read-across” or grouping), which are classified as corrosive to the skin (Skin Corrosive Cat. 1), or do suitable (Q)SAR methods indicate corrosion potential of the substance?	NO	Predicted to be "Not Irritating or Corrosive to Skin" using Toxtree (v2.6.6). . The outcomes of OASIS TIMES v2.27.17 does not support the substance to be corrosive neither (the model predicted the substance to be Irritating to skin >> Ethers but not , with 87.5%of correct fragment).
	5b	Are there structurally related substances (suitable “read-across” or grouping), which are classified as irritant to the skin (Skin Irritant Cat. 2), or indicating that the substance is non-irritant, or do suitable (Q)SAR methods indicate irritant or non-irritant potential of the substance?	NO
Existing in vitro data	6a	Has the substance demonstrated corrosive properties in an EU/OECD adopted in vitro test? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met	NO
	6b	Has the substance demonstrated irritant or non-irritant properties in an EU/OECD adopted in vitro test? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.	NO	(at the initiation of the dossier, no test was available)
	6c	Are there data from a non-validated suitable in vitro test(s), which provide sound conclusive evidence that the substance is corrosive/ irritant?	NO
Weight-of- Evidence analysis	7	The “elements” described above may be arranged as appropriate. Taking all available existing and relevant data mentioned above (Elements 1-6) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and –if so –how to classify and label?	NO
New in vitro test for corrosivity	8	Does the substance demonstrate corrosive properties in (an) EU/OECD adopted in vitro test(s) for skin corrosion?	NO	Based on all available data (ATD, LLNA, QSARs), the substance is not considered as a skin corrosive => a skin corrosion assay was not required
New in vitro test for irritation	9	Does the substance demonstrate irritating or non-irritating properties in (an) EU/OECD adopted in vitro test(s) for skin irritation?	YES	=> an Episkin test for irritation was initiated. The conclusion of this Episkin test is sufficient to conclude on C&L (viability = 97,3% <=> Not a skin irritant)
New in vivo test for corrosion/irritation	10	To be used only as a last resort	NO	In vivo testing should not be conducted in this case since the substance falls under the scope of the specific in vitro tests performed, and there are no substance-specific limitations on use of those tests. An adaptation according to Annex XI to the REACH Regulation is included in this dossier.

The purpose of the newly performed in vitro test (HLS, 2015, Rel.1) was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours.

This test was performed in compliance with GLP. The quality criteria required for acceptance of results in the test were satisfied. The relative mean viability of the test item-treated tissues was 97.3 ± 10.7 %, after the 15‑minute exposure period. With a tissue viability > 50%, the registered substance was considered to be non-irritating to skin.

Eye irritation:

A key study was identified (FREY-TOX, 2000, rel.1). In this in vivo eye irritation study performed according to the OECD guideline No. 405, and in compliance with GLP, 0.1 mL of undiluted test material was instilled into one eye of 4 female New Zealand White rabbits. The other eye remained untreated and served as control. The upper and lower eyelids were held together for about one second immediately after instillation. The eyes were examined and the changes were observed at 1, 24, 48 and 72 h after treatment and graded according to the Draize method.

The calculated mean score for each animal within 3 scoring times (24, 48 and 72 h) were 0.33/0.0/0.67/0.33 for redness, 0/0/0/0 for chemosis, 0/0/0/0 for iris lesions and 0/0/0/0 for corneal opacity. The effects observed were all reversible within 72 hours. The substance is therefore not classified for eye irritation.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data :

- no additional self-classification is proposed for the registered substance regarding skin irritation according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP)

- the registered substance is classified as Category 3 according to the GHS based on slight irritation observed in human patch test and in acute dermal toxicity study.

Based on the available data no additional self-classification is proposed regarding eye irritation according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

No data was available regarding respiratory irritation, however the substance not being classified for skin and eye irritation according to CLP criteria, no classification is expected for respiratory irritation.