4-allylveratrole

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

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. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-allylveratrole

EC Number:

202-223-0

EC Name:

4-allylveratrole

Cas Number:

93-15-2

Molecular formula:

C11H14O2

IUPAC Name:

1,2-dimethoxy-4-(prop-2-en-1-yl)benzene

Test material form:

liquid

Test animals / tissue source

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.

Test system

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

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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	I
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.

Applicant's summary and conclusion

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.