Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 June 2021 to 24 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction

EC Number:

953-265-8

Molecular formula:

Not applicable

IUPAC Name:

Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ model and the corresponding test method have been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline (OECD No. 492), and the method is applicable to mixtures, solids, liquids, semi-solids and waxes. Therefore, it was considered to be suitable for this study.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The test was performed using EpiOcular™ Reconstructed Human Corneum Epithelium (RhCE) from MatTek Corporation. The system consists of normal, human-derived corneal 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. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2. The certificate of Analysis of the test system is included in the report. The tissue viability and barrier function tests are within the acceptable ranges and indicate the appropiate formation of the mucosal barrier and a viable basal cell layer. Tissues were used within 48 h of their delivery.
The cells used to produce EpiOcular (TM) tissue are screened for potential biological contaminants:
- HIV-1 virus - oligonucleotide-directed amplification: Not detected.
- Hepatitis B virus - oligonucleotide-directed amplification: Not detected.
- Hepatitis C virus - oligonucleotide-directed amplification: Not detected.
- Bacteria, yeast and other fungi - long term antibiotic, antimycotic free culture: Not detected.

- RhCE tissue or hCE cell construct used, including batch number: 2 living RhCE tissue replicates (EpiOcularTM OCL-200, supplied by MatTek Corporation, batch No. 34916) and 2 additional killed Human skin models (EpiOcularTM OCL-200, supplied by MatTek Corporation, batch No. 34911).

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): 50 mg for each replicate.

Duration of treatment / exposure:

5 hours and 47 minutes

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2 tissues for the test substance, 2 additional tissues for NSMTT and 2 tissues for each control.

Details on study design:

- Details of the test procedure used: Two tissues (0.6 cm2) were pre-wetted with 20 µL DPBS buffer, incubated for 32 min, then dosed topically with 50 mg test item and exposed for 5 hours and 47 min at 37ºC, 5% CO2, ≥ 95% RH. After exposure, the tissues were rinsed with DPBS, transferred to fresh assay medium, and incubated for 25 min. Then, they were transferred to fresh medium again and incubated for 18 hours at 37ºC, 5% CO2, ≥ 95% RH.
The MTT assay for cell viability evaluation was performed on the tissues, by incubating them for 3 hours with MTT solution at 37ºC, 5% CO2, ≥ 95% RH. The precipitated formazan crystals were then extracted using isopropanol and quantified spectrophotometrically.
- Doses of test chemical and control substances used: 50 mg test item, 50 µL in controls.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: exposure 5 h and 47 min at 37ºC, 5% CO2; post-exposure immersion 25 min at room temperature; post-exposure incubation 18 h at 37ºC, 5% CO2.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): the test item was identified as a direct MTT reducer. Thus, 2 killed control tissue models were added to the study which were treated in the same manner that the living ones in order to generate non-specific MTT reduction.
- Number of tissue replicates used per test chemical and controls: 2 tissues for the test substance, 2 additional tissues for NSMTT and 2 tissues for each control.
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device: Wavelength: 570 nm.
- Description of the method used to quantify MTT formazan: Optical density by microtiter plate photometer.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: According to OECD 492, the percentage tissue viability cut-off value distinguishing classified from non-classified test chemicals is 60%. Therefore, the test item is identified as not requiring classification if the mean percent tissue viability after exposure and post-exposure incubation is > 60%. Otherwise, the test item is identified as potentially requiring classification and labelling.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: yes, included in the study report.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The validity of the RhCE EpiOcular™ test at laboratory facility was demonstrated in a proficiency study. For this purpose 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized.
- Positive and negative control means and acceptance ranges based on historical data: yes, see table below.
- Acceptable variability between tissue replicates for positive and negative controls: yes, 2.8% (negative control) and 0.9% (positive control). The values for negative and positive controls were below 20% (OECD Guideline 492).
- Acceptable variability between tissue replicates for the test chemical: yes, 1.5% and 13% (NSMTT control), below 20% (OECD Guideline 492).

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

mean percent tissue viability 

Run / experiment:

test item

Value:

99.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

mean percent tissue viability 

Run / experiment:

NSMTT

Value:

40.13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Test item corrected = test item viability - NSMTT viability

Value:

59.35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (mean OD = 0.866; As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range > 0.4 and < 1.4 for the negative control)
- Acceptance criteria met for positive control: yes (mean cell viability = 30.1%; demanded < 50% of negative control)

Any other information on results incl. tables

Table 1: Assessment of the eye irritation potential individual and average values of OD

	Well ID	OD	Mean OD  / disc ( #)	Mean OD / product	Viability %	Mean viability %	Difference of viability	Conclusion
Negative control	SPL1	0,678 1,089 0,794	0,854	0,866	98,6	100,0	2,8
	SPL2	0,820 0,892 0,922	0,878		101,4
Positive control	SPL3	0,241 0,260 0,269	0,257	0,261	29,7	30,1	0,9	UN GHS Category 2 or 1
	SPL4	0,247 0,273 0,273	0,265		30,6
Test item PH-21/0194	SPL7	0,810 0,855 0,900	0,855	0,862	98,7	99,5	1,5	UN GHS Category 2 or 1
	SPL8	0,765 0,904 0,933	0,868		100,2
Test item PH-21/0194 NSMTT	SPL9	0,271 0,296 0,305	0,291	0,348	33,6	40,13	13,0
	SPL10	0,375 0,418 0,419	0,404		46,7
Test item PH-21/0194 corrected						59,35		UN GHS Category 2 or 1

#: mean of 3 values (triplicate of the same extract)

OD: optical density

SPL: sample

Acceptability criteria:

Tissues treated with the positive control substance should show a mean tissue viability < 50%.

The difference of viability between two tissue replicates should be less than 20%.

Negative control: OD values of the two replicates should be in the range > 0.8 and < 8. As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range > 0.4 and < 1.4 for the negative control

 

Applicant's summary and conclusion

Interpretation of results:

other: the results obtained under these experimental conditions lead to the category “no prediction can be made” and further testing is required to establish a definitive classification

Conclusions:

Under the conditions of the test, no prediction can be made as the test substance is considered either eye irritant or inducing serious eye damage in the EpiOcular™ Eye Irritation Test.

Executive summary:

An in vitro eye irritation test (EIT) according to OECD TG 492 was conducted under GLP conditions to assess the irritation potential of the test item in reconstructed human cornea-like epithelium (RhCE) tissues (EpiOcular^TM tissue model). Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. The test item was identified as a direct MTT reducer, hence 2 additional killed control tissues were added to the study and treated in the same manner that the living tissues. 2 tissues (0.6 cm2) were pre-wetted with 20 µL DPBS buffer, incubated for 32 min, then dosed topically with 50 mg test item and exposed for 5 hours and 47 min at 37ºC, 5% CO2, ≥ 95% RH. After exposure, the tissues were rinsed with DPBS, transferred to fresh assay medium, and incubated for 25 min. Then, they were transferred to fresh medium again and incubated for 18 hours at 37ºC, 5% CO2, ≥ 95% RH. The MTT assay for cell viability evaluation was performed on the tissues, by incubating them for 3 hours with MTT solution at 37ºC, 5% CO2, ≥ 95% RH. The precipitated formazan crystals were then extracted using isopropanol and quantified spectrophotometrically (OD at 570 nm) in order to determine the cell viability. Concurrent positive and negative controls were run in parallel. All validity criteria were met. The relative true viability (that is, mean test item viability % - mean NSMTT control viability %) of the tissues treated with the test item was 59.35%. This value is below the threshold for eye irritation potential (≤ 60%) which means that the test item is considered either eye irritant or inducing serious eye damage. Thus, no prediction can be made.