Resinoid of Liquidambar styraciflua (Hamamelidaceae) obtained from exudate by organic solvents extraction

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September to 21 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 492 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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)

Version / remarks:

July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

29 June 2015

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

German GLP Compliance Program (inspected on July 13-16, 2015 / Signed on September, 2015)

Test material

Test material information

Constituent 1

Reference substance name:

Resinoid of Liquidambar styraciflua (Hamamelidaceae) obtained from exudate by organic solvents extraction

EC Number:

947-390-7

IUPAC Name:

Resinoid of Liquidambar styraciflua (Hamamelidaceae) obtained from exudate by organic solvents extraction

Test material form:

liquid: viscous

Details on test material:

- Appearance: Yellow to pale brown very viscous liquid

Specific details on test material used for the study:

Storage Conditions: In the refrigerator, under N2 atmosphere, under light protection, under moisture protection

Test animals / tissue source

Species:

other: human reconstructed cornea model (EpiOcular™)

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. This test guideline is applicable to solids, liquids, semi-solids and waxes, so is considered to be applicable to the test item.

- CELL SYSTEM:
- EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (Ashland, MA01721, USA).
- Lot No.: 27005
- Keratinocyte strain: 4F1188
- Analysis for potential biological contaminants: none detected (HIV-1, Hep. B and Hep. C virus; Bacteria, yeast and other fungi)
- Tissue viability: 1.419, within the acceptance criteria (1.1-3.0)
- Barrier function: 15.97 min, within the acceptance criteria (12.2-37.5)
- Sterility: Sterile

- The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL, 10 mm of diameter).
- Transport: EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate.
- Storage: On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. An appropriate volume of EpiOcular™ Assay Medium was warmed to approximately 37 °C. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (nearly 17 hours).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The test item was warmed in a water bath at 37° C to be better pipettable. 50 µL of the test item were dispensed directly atop duplicate EpiOcular™ tissue.

Duration of treatment / exposure:

30 minutes

Observation period (in vivo):

NA

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

duplicate tissues

Details on study design:

- Details of the test procedure used: procedure for solids
- RhCE tissue construct used, including batch number: EpiOcular™ kits (Lot No.: 27005)
- Doses of test chemical and control substances used: 50 µL
- Duration and temperature of:
exposure: 30 minutes / 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
post-exposure immersion: 12 min / room temperature
post-exposure incubation period: 120 minutes / 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
- Description of any modifications to the test procedure: none
- Test for direct MTT reduction: 50 µL of the test item are added to 1 mL of a 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 h. Untreated MTT solution was used as a control. Then the colour of the obtained solutions was evaluated.
- Assessment of Color Interference with the MTT endpoint: 50 µL of the test item are added to 2 mL of isopropanol and to 1 mL of water. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for one hour. The isopropanol mixture was left for 3 hours at room temperature..
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT): 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The results are acceptable according to OECD TG 492, if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant (no Category according to UN GHS).
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant (Category 2 or Category 1 according to UN GHS; no differentiation between the categories possible).
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Data of 30 studies performed from July 2015 until middle of November 2016
Positive controls: Viability 32.3% (6.90-43.4) / Absorption 0.566 (0.107-0.943)
Negative controls: Absorption 1.65 (1.27-2.16)
- Complete supporting information for the specific RhCE tissue construct used: yes, attached to the study report
- Reference to historical data of the RhCE tissue construct: no
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: yes
- Positive and negative control means and acceptance ranges based on historical data: yes
- Acceptable variability between tissue replicates for positive and negative controls: yes
- Acceptable variability between tissue replicates for the test chemical: yes

Results and discussion

In vitro

Results

Irritation parameter:

other: tissue viability

Run / experiment:

Mean

Value:

96.8

Vehicle controls validity:

valid

Remarks:

Not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

36.3%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item proved to be a MTT reducer in the MTT pre-test. An additional test with freeze-killed tissues was preformed to evaluate whether the test material is not binding to the tissue and leading to a false MTT reduction signal.

OTHER EFFECTS:
- Visible damage on test system: none reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The negative control OD is > 0.8 and < 2.5 (values between 1.616 and 1.636).
- Acceptance criteria met for positive control: yes. The mean relative viability of the positive control is below 50% of the negative control viability (36.3%).
- Acceptable variability between tissue replicates: yes. The difference of viability between the two relating tissues of a single item is < 20% in the same run (values between 0.3% and 3.7% for positive and negative control treated tissues as well as test item treated tissues and killed controls).

Any other information on results incl. tables

Table 7.3.2/1: Results after 30 minutes treatment with the test item and the controls

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected		Mean OD of Treatment Group blank corrected	Rel. Viability [%] Tissue 1, 2 *	Absolute Value of the Difference of Rel. Viability  Tissue 1,2[%]	Mean Rel. Viability [%]**
Blank		0.036	0.036	0.036
Negative Control	1	1.628	1.644	1.636	1.601		1.590	100.6	1.3	100.0
	2	1.617	1.614	1.616	1.580			99.4
Positive Control	1	0.585	0.635	0.610	0.574		0.577	36.1	0.3	36.3
	2	0.618	0.613	0.615	0.580			36.4
Test Item	1	1.669	1.659	1.664	1.628		1.657	102.4	3.7	96.8***
	2	1.719	1.725	1.722	1.686			106.0
Blank		0.036	0.036	0.036
Negative Control Freeze killed Tissues	1	0.098	0.104	0.101	0.065		0.068	4.1	0.4	4.3
	2	0.107	0.107	0.107	0.071			4.5
Test Item Freeze killed Tissues	1	0.230	0.233	0.232	0.196		0.185	12.3	1.3	11.7
	2	0.211	0.210	0.210	0.175			11.0

*                            Relative absorbance [rounded values]:

 

**                          Mean relative absorbance [rounded values]:

***            KC corrected test item viability = mean viability_{test item}– mean viability_TestItemKC= 104.2% - 7.4% = 96.8%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study and with a percentage of tissue viability > 60%, the test item does not require classification for eye irritation or eye damage according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

A study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The study was conducted according to the OECD guideline No. 492 and in compliance with GLP.

The test item proved to be an MTT reducer in the MTT pre-test. An additional test with freeze-killed tissues was preformed to determine a correction factor for calculating the true viability in the main experiment.

50 µl of the test item was applied to each of duplicate tissue for 30 minutes. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

Treatment with the positive control induced a decrease below 50% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system.

The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues.

The quality criteria required for acceptance of results in the test were satisfied. The tissue viability of the test item was 96.8% after the 30 -minute exposure period.

Under the experimental conditions of this study and with a percentage of tissue viability > 60%, the test item does not require classification for eye irritation or eye damage according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

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