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Description of key information

4 -allylveratrole/Methyl Eugenol was tested for its possible skin irritation potential using a three dimestional Reconstructed Human Epidermis model, EpiSkin according to OECD 439 guideline. The tissue viability met the acceptance criterion. Mean OD570of negative control was  0,646587. The viability of culture treated by positive control 5% SDS was 6.9%. The positive control met the acceptance criterion: mean tissue viability less than 20%. Determined viability of culture treated by 4 -allylveratrole/Methyl Eugenol (123 %) fulfilled the criteria for non-irritancy.

4 -allylveratrole, was tested for its possible skin corrosion potential using a three dimensional Reconstructed Human Epidermis model, EpiSkin according to OECD 431 guideline. The absolute mean OD570of the negative control tissues was 0.55533, 0.67628 and 0.45058 for the 3 -minute, 60 -minute and 240 -minute exposures, respectively. The positive control has a mean cell viability of 8.95% after 240 -minute exposure. Thus, the results of the controls indicate that the test system functioned properly.

After 240 -minute exposure the test item showed tissue viability 177 %. Taken together, the test item 4 -allylveratrole is non-corrosive in the in vitro skin corrosion test.

In vitro eye irritation test was carried out using EpiOcularTM Human tissue model with an objective to evaluateEye Irritation potential of the test item 4 -allylveratrole. The study was conducted according OECD 492 guideline and fulfilled the acceptance criteria. As the test item-treated tissues showed viability of >60.0% relative to negative control-treated tissue viability, 4 -allylveratrole was concluded to be non-irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 October 2017-23 November 2017
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes
Test system:
human skin model
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE

EpiSkin Small Model kit provided by SkinEthic

- Model used: EpiDerm™ (EPI-200-SIT)
- Tissue batch number(s): 17-EKIN-044
- Delivery date: 31 October , 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: incubator 37±1°C
- Temperature of post-treatment incubation (if applicable): incubator 37±1°C, 5±1% CO2



Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 ul
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
Three
Details on study design:
The test item was tested for possible skin irritation potentiial using RHE model, EpiSkin through topical application for 15 minutes. After 42 hour post-incubation perod, irritation potential of the test item was evaluated by assessing cytotoxic (irritancy) effect. Cytotoxicity is expressed as a reduction of mitochodrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
Irritation / corrosion parameter:
% tissue viability
Value:
122.59
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 1. Skin irritation potential of 4 -allylveratrole after 15 min exposure in reconstructed human epidermis (RHe)

 Sample  OD Mean  SD of OD  Viability mean %  In vivo prediction
 Negative control  0,646587  0,006166  100  NI
 Positive control  0,03837  0,001103 5,93   I
 Test item  0,79262  0,001477  122,59  NI

OD= optical density

S=standard deviation

NI=non-irritant

I=irritant

Interpretation of results:
GHS criteria not met
Conclusions:
The test item, Methyl Eugenol/4-allylveratrole, is non-irritant in the in vitro skin irritation test using reconstructed human epidermis.
Executive summary:

The test item 4 -allylveratrole/Methyl Eugenol was tested for its possible skin irritation potential using a three dimestional Reconstructed Human Epidermis model, EpiSkin. The magnitude of viability was quantified by using MTT test. Validity of the test method was ascertained by positive control 5% SDS. Three tissue replicates were used for each treatment (exposure time 15 minutes), including negative and positive control.  

The tissue viability met the acceptance criterion. Mean OD570 of negative control was  0,646587. The viability of culture treated by positive control 5% SDS was 6.9%. The positive control met the acceptance criterion: mean tissue viability less than 20%. Determined viability of culture treated by 4 -allylveratrole/Methyl Eugenol (122,59%) fulfilled the criteria for non-irritancy. Therefore, the is considered to be non-irritant to the skin.

Criteria for interpretation

Classification UN GHS

Mean tissue viability is50%

Category 2 or Category 1

Mean tissue viability > 50%

Non-Irritant

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 August 2017- 22 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
EpiSkin
Vehicle:
unchanged (no vehicle)
Details on test system:
Source of the test system: SkinEthic Laboratories, France.
Soon after reciept, the epidermis units were transferred to 12-well plates containing 2ml of pre-warmed medium and incubated in CO2 incubator for 43 hours.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 ul
Duration of treatment / exposure:
3 min, 60 min and 240 min
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minutes exposure
Value:
173.15
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60-min exposure
Value:
129.94
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
240-min exposure
Value:
176.58
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
8.95

OD values of individual epidermis units.

Table 1. 3 -min exposure

      Absorption (OD570)
   R1  R2
 Negative control

 0,5604

0,55025 

 Test item

 0,95955

0,9635

Table 2. 60 -minutes exposure

           Absorption (OD570)
   R1  R2
 Negative control  0,66795 0,6846 
 Test item  0,771 0,9862

Table 3. 240 -minutes exposure

       Absorption (OD570)  
   R1  R2
Negative control  0,4484 0,4575 
 Test item  0,79745  0,79385
 Positive control  0,04195  0,03865
Interpretation of results:
GHS criteria not met
Conclusions:
Since the tissue viability is > 35% after the 240 minute exposure, the test item 4-allyveratrole is non-corrosive under the experimental conditions described.
Executive summary:

The test item, 4 -allylveratrole, was tested for its possible skin corrosion potential using a three dimesnsional Reconstructed Human Epidermis model, EpiSkin, through topical application. The absolute mean OD570 of the negative control tissues was 0.55533, 0.67628 and 0.45058 for the 3 -minute, 60 -minute and 240 -minute exposures, respectively. The positive control has a mean cell viability of 8.95% after 240 -minute exposure. Thus, the results of the controls indicate that the test system functioned properly.

After 240 -minute exposure the test item showed tissue viability 177 %. Taken together, the test item 4 -allylveratrole is non-corrosive in the in vitro skin corrosion test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
Study initiation date: 18 Septermber 2017. Study completion date: 30 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals or tissues and environmental conditions:
EpiOcular TM Tissue from MatTek, lot number 27005. Keraticocyte strain 4F1188.
Tissues were incubated at 37±1oC, 5% CO2 and humidified atmosphere.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 ul
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
120+-15 minutes
Number of animals or in vitro replicates:
Two tissues, two aliquots
Details on study design:
Assessment of Direct MTT reduction by the Test Item
A quantity of 50 μL of liquid test item was added to 1 mL of MTT solution (1.0 mg / mL) in a 6 well plate and incubated in dark at standard culture conditions for 3 hours. Concurrent negative control (50 μL sterile deionized
water) was tested along with test item. Post incubation, test item did not convert the MTT solution to blue or purple color, indicates test item is not reducing the MTT solution.

Assessment of Colored or Staining Materials
Colored test item or test items which become colored after application to the tissues may interfere with the quantitative MTT measurement. Therefore, the test items are checked for its colorant properties.
In this context, chemicals which absorb light and appear red, yellow, green, and blue dark purple and black are considered as intrinsic colorants.
a. Blue, dark purple and black test items: As the test item is not colored,test was not performed.
b. Other intrinsically colored test items (e.g. red, yellow, green colorants): A volume of 50 μL test item was added to 2 mL of isopropanol, incubated in 6-well plates, and placed on an orbital plate shaker for 2 hours and 50 minutes at room temperature. Two 200 μL aliquots of isopropanol solutions (mixed with test item) and of pure isopropanol was transferred to a 96-well plate and the absorbance was measured with a plate reader and tested for their ability to absorb significantly light at 570 nm (wavelength used for the MTT determination). 200 μL aliquots of the mixture was used for measurement and measured the OD at 570 nm.
After subtraction of the OD for isopropanol, the OD of the test item solution is not > 0.08 (approximately 5% of the mean viability of the negative control), hence test item is considered as non-interactive with the
MTT measurement and an additional test on colorant controls (CC) was not performed.
c. Non coloured test item: As the test item is non-colored after contact with water or isopropanol, it was considered as non-interactive with the MTT. Measurement and an additional test on colorant controls (CC) were not
performed.

Preparation of EpiOcular Tissues for Treatment
On the day of tissue receipt (19 September 2017) the tissues in its 24-well plate shipping container, were equilibrated to room temperature for about 15 minutes. EpiOcular™ assay medium (1 mL/well) was added to 6 well plates and warmed to approximately 37ºC in a CO2 incubator. Each 12 well shipping containers were removed from the plastic bag under sterile conditions and surface disinfected by wiping with 70% ethanol. Each tissue in the 12 well plates was inspected for air bubbles between the insert and the agarose gel. The tissues were removed carefully from the 12 well plate using sterile forceps, agarose adhering to insert was removed gently by blotting on to sterile filter paper and placed in the 6 well plate containing 1 mL of EpiOcular™ assay solution for 1 hour. After 1 hour, assay medium was replaced with fresh assay medium and incubated further for 16 hours at standard culture conditions
Irritation parameter:
in vitro irritation score
Run / experiment:
The mean tissue viability (%)
Value:
144.08
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100.0
Positive controls validity:
valid
Remarks:
30.75

Table 1. OD values of individual epiocular tissues.

      Tissue 1     Tissue 2
  Sample  R1  R2  R1  R2
 Negative control  1.3252  1.2966  0.9017  0.8696
 Positive control  0.3901 1.6199  1.5355  1.5177
 Test item  1.5845  1.6199  1.5355  1.5177

Table 2. The percentage viablity of the epiocular tissues.

 

Viability Tissue 1       

       Viability Tissue 2      
 Sample  Ali 1 Ali 2 Mean 1 Ali 1   Ali 2  Mean Mean 1&2 Diff 1 & 2   Classification
 NC 121,46 118,75 120,11 81,41 78,38 79,89 100,00  40,21  NI 
 PC  33,03 31,24  32,13  28,95  29,80  29,37  30,75  2,76 
 Test item  145,98 149,33 147,65  141,35  139,66  140,50   144,08 7,15  NI 

NC: Negative control (deionized water), PC: Positive control (methyl acetate), I: Irritant, NI:Non-irritant, Ali: Aliquot, Diff: difference of viability between two tissues.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item showed viability of >60% relative to negative control-treated viability. Thus, the test item, 4-allylveratrole, was concluded to be non-irritant in the EpiOcularTM test.
Executive summary:

A guideline compliant eye irritation study (OECD 492) –in vitro eye irritation in Reconstructed Human Cornea-like Epithelium (RhCE)) was conducted. Aim of the study was to examine the acute eye irritation potential of the test item by measurement of its cytotoxic effect on the EpiOcular™ cornea epithelial model. The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek).

 

In this assay, the test item was applied to the surface of the cornea epithelial construct for 30 minutes. After exposure period, each tissue was extensively rinsed, and the tissue was allowed to express the resulting damage. Two construct tissues were used for the treatment with test item and for negative and positive controls. The MTT assay was performed to determine tissue viability. The tissues were transferred in the 24-wells plate containing MTT medium and incubated in a humidified atmosphere for 3 hours. After incubation the blue formazan salt formed was extracted in isopropanol and the optical density of extracted formazan was determined using a spectrophotometer at 540 nm. Validity of the test method was ascertained by positive control (methyl acetate) and negative control (deionized sterile water). The tissue viability met the acceptance criterion as the mean OD of negative controlwas in between > 0.8 and < 2.5. The positive control met the acceptance criterion as the viability of culture treated by methyl acetate was 30.8% (less than 60%).The viability of culture treated by 4-allylveratrole was 144.1%. Therefore, 4-allylveratrole is considered to be non-irritant to the eye.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification

The results of the key studies (OECD439 & 431) do not indicate the substance to be classified for skin irritant according to CLP Regulation 1272/2008.

The result of the key study (OECD492) does not indicate the substance to be classified for eye irritant according to CLP Regulation 1272/2008.