Balmywood

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 January 2022 to 17 February 2022.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 and under GLP compliance (the exact composition of the test item cannot be exactly determined because the element is an UVCB substance. This deviation to GLP is under the responsibility of the Sponsor and is considered as without impact on the conclusion of the study).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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)

Version / remarks:

Adopted 18 June 2019

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Light fraction extract obtained from the labdanum gum issued from the exudate of Cistus ladaniferus (Cistaceae) by maceration with essential oil of Cedarwood obtained from the wood of Juniperus Mexicana (Cupressaceae)

Molecular formula:

not applicable for NCS substances

IUPAC Name:

Light fraction extract obtained from the labdanum gum issued from the exudate of Cistus ladaniferus (Cistaceae) by maceration with essential oil of Cedarwood obtained from the wood of Juniperus Mexicana (Cupressaceae)

Test material form:

liquid

Test animals / tissue source

Species:

human

Strain:

other: Keratinocyte strain No.4F1188

Details on test animals or tissues and environmental conditions:

CELL SYSTEM USED
0.60 cm² Reconstructed human Cornea-like Epithelia (EpiOcular™ OCL-200-EACH, supplied by MatTek Corporation, batch No. 34953) containing normal human keratinocytes.

ANALYSIS FOR POTENTIAL BIOLOGICAL CONTAMINANTS
The cells used to produce EpiOcular™ tissue are screened for potential biological contaminants.
HIV1 virus, hepatitis B virus and hepatitis C virus were checked using oligonucleotide-directed amplification and bacteria, yeast and other fungi were checked using long term antibiotic, antimycotic free culture.
> No contamination was detected for all contaminants.

ANALYSIS FOR TISSUE FUNCTIONALITY AND QUALITY
- The tissue viability was checked using a MTT QC assay for 1hour. The OD value (540-570nm) should be between 1.1 and 3. As the OD value = 1.783±0.055, the cell viability is confirmed.
- The barrier function represented by the Exposure Time inducing 50% viability (ET50) was checked using Triton X-100 and should be between 12.2 and 37.5 minutes. As the ET50= 28.17 minutes, the barrier function is considered valid.

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 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)

Duration of post- treatment incubation (in vitro):

- Post-exposure immersion period: 12 minutes at room temperature - Post-exposure incubation period: 2 hours at standard culture conditions

Number of animals or in vitro replicates:

Test item, negative and positive controls were applied to the entire surface of 4 living RhCE tissue replicates including 2 RHE models for the non-specific colour (NSC) control.

Details on study design:

TISSUES CONSTRUCTS
- RhCE tissue construct used: 0.60 cm² Reconstructed human Cornea-like Epithelia (EpiOcular™ OCL-200-EACH, supplied by MatTek Corporation, batch No. 34953) were received on 15 February 2022.

- Pre-incubation of the tissues:
On 15 February 2022, the tissues in their well shipping container were equilibrated to room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium (MatTek Corporation, batch No. 021422MPA) and incubated for 22 hours and 02 minutes at standard culture conditions.

- Indication of controls used for direct MTT-reducers:
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of solution of MTT at 1 mg/mL (same conditions as in the main test). A yellow liquid was observed after 3 hours of incubation between 36.5°C and 36.9°C, 5% CO2.
> Therefore, there is no direct interaction between the test item and MTT and there is no need to add non-specific MTT reduction (NSMTT) controls.

- Indication of controls used for colouring test chemicals:
The spectral properties at 570 nm of test item in isopropanol were checked by adding 50 µL of the test item to 2 mL of isopropanol (same conditions as in the main test). An orange solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.114 which is higher than 0.08 (value corresponding to approximately twice the OD of the extracting solvent).
> Therefore, the test item was identified as causing colour interference with the viability assay and two viable control tissues were added to the study which underwent the entire testing procedure but were incubated with culture medium instead of MTT solution during the MTT incubation step to generate a non-specific colour (NSC living) control.

MAIN TEST
- Pre-treatment:
After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 3730921). The tissues were incubated at standard culture conditions for 30 minutes.

- Treatment and post-treatment incubation of the tissues:
The test item was applied as supplied, at the dose of 50 μL, to the entire surface of 4 living RhCE tissue replicates including 2 RHE models for the non-specific colour (NSC) control, during 30 minutes at standard culture conditions.

In the same experimental conditions, a positive control (Methyl acetate - Sigma-Aldrich, batch No. BCBX8836) and a negative control (distilled water - ADL Prochilab - Batch No. 2011117) were carried out. The controls were applied, as supplied, at the dose of 50 μL, to the surface of 2 RhCE tissue replicates during 30 minutes at standard culture conditions.

After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 3730921). The rinsed tissues were checked for any coloration and noted to be white, comparable coloration to that of the negative control tissues, with residual brown coloration in the center.
This rinsing step was followed by a 12-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue.
The RhCE constructs were then incubated for a 2 hours post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.

- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at standard culture conditions. The two additional control tissues were incubated in assay medium (MatTek Corporation, batch No. 021422MPA) instead of MTT solution in order to generate NSC control.

The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol for 16 hours and 40 minutes at 7+/-3°C in the dark.
The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2).

The OD at 570 nm was measured in triplicate samples of formazan extracts.
The measured OD are proportional to the number of living cells.
The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Results and discussion

In vitro

Results

Irritation parameter:

other: % mean corrected percent tissue viability

Run / experiment:

After 30 minutes exposure

Value:

104

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:

MTT VIABILITY ASSAY RESULTS
As the test item was identified as causing colour interference with the viability assay, the true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the interfering test chemical and incubated with the MTT solution (%Viability test) minus the percent non-specific colour obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT, run concurrently to the test being corrected (%NSC living).

> Therefore, the corrected mean percent viability of the RhCE replicates treated with the test item BALMYWOOD ZA3155 was 104.0% (considered as 100%) versus 48.4% in the positive control (Methyl acetate).

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 492.

ACCEPTANCE OF RESULTS:
These results are in accordance with the acceptability criteria:
- The OD of the negative control was > 0.8 and < 2.8. 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 (mean OD of the negative control = 0.793);
- The relative mean tissue viability for the positive control treated tissues was <50% relative to the negative control treated tissues (mean viability of the positive control = 48.4%);
- The difference of viability between two tissue replicates is < 20% (difference of viability between two tissue replicates is 7.9% and 0.4% for living tissues exposed to the test item and NSC living tissues exposed to the test item and then incubated in medium instead of MTT, respectively).

Any other information on results incl. tables

Table 7.3.2/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls after 30 minutes exposure

 

Tissue	OD	Mean OD/ disc (#)	Mean OD / Product	Viability (%)	Mean viability (%)	SD viability	Viability difference between replicates (%)	Conclusion
Negative Control	0.743 0.816 0.800	0.786	0.793	99.1	100.00	1.2	1.8	No category
	0.769 0.853 0.778	0.800		100.9
Positive Control	0.350 0.366 0.366	0.361	0.384	45.5	48.4	4.1	5.8	UN GHS Category 2 or 1
	0.394 0.417 0.409	0.407		51.3
Test Item	0.782 0.828 0.787	0.799	0.831	100.8	104.7	5.6	7.9	No category
	0.843 0.898 0.846	0.862		108.7
Test item NSC living	0.009 0.007 0.006	0.007	0.006	0.9	0.7	0.3	0.4
	0.004 0.004 0.004	0.004		0.5
Test item corrected					104.0

#: mean of 3 values

OD: optical density

SPL: sample

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The corrected mean percent viability of the RhCE replicates treated with the test item BALMYWOOD ZA3155 was 104.0% (considered as 100%) versus 48.4% in the positive control (Methyl acetate).
Under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and the signal word are required.

Executive summary:

An OECD 492 study was performed to evaluate the eye hazard potential of test item after topical administration on in vitro reconstructed human cornea-like epithelium tissues (EpiOcular™ tissue model).

 

The test item BALMYWOOD ZA3155 was applied as supplied, at the dose of 50 µL, to 2 living DPBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). In the same experimental conditions, a positive control (Methyl acetate) and a negative control (distilled water) were carried out. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 RhCE tissue replicates during 30 minutes at standard culture conditions.

The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay. Additionally, 2 RhCE (EpiOcular™ tissue model) were treated in the same manner but were incubated in culture medium instead of MTT solution in-order-to generate non-specific living color controls.

 

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

The mean corrected percent tissue viability of the RhCE replicates treated with the test item BALMYWOOD ZA3155 was 104.0% (considered as 100%) versus 48.4% in the positive control (Methyl acetate).

  

In conclusion, under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item BALMYWOOD ZA3155 does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and the signal word are required.