3,4-dihydroxybenzaldehyde

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09-08-2022 to 25-08-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydroxybenzaldehyde

EC Number:

205-377-7

EC Name:

3,4-dihydroxybenzaldehyde

Cas Number:

139-85-5

Molecular formula:

C7H6O3

IUPAC Name:

3,4-dihydroxybenzaldehyde

Test material form:

solid

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Description of the cell system used: EpiOcular TM
- RhCE tissue or hCE cell construct used, including batch number: 0.60 cm2 reconstructed human Cornea-like Epithelium. supplied by MatTek Corporation, batch No. 34980.
- Tissues in their shipping container were equilibrated to room temperature for 20 minutes, then were removed from agarose and placed in 6 wells culture plate which had been filled with 1 mL of 37º pre-warmed assay medium and incubated 20 hours and 25 minutes at standard culture conditions.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Positive and negative control dose: 50 μL
- Test item: 50 mg

Duration of treatment / exposure:

- Positive and negative control dose: 50 μL: applied to the surface of 2 RhCE tissue replicates for 5 hours and 55 minutes.
- Test item: 50 mg: applied to the surface of 2 RhCE tissue replicates for 5 hours and 55 minutes.

Duration of post- treatment incubation (in vitro):

17 hour and 45 minutes post exposure incubation

Number of animals or in vitro replicates:

4 RhCE tissue replicates

Details on study design:

TREATMENT, POST-IMMERSION AND POST-INCUBATION:
- Pre-treatment: tissues were pre-wetted with 20 μL of Ca2+, Mg2+ Free-DPBS and incubated for 30 minutes.
- Treatment: Test item (dose 50 mg) was applied to 2 RhCE tissue (5 hours, 55 minutes); positive and negative control were applied (50 μL) to 2 RhCE tissue (5 hous and 55 minutes).
- Post exposure incubation period: substances were washed from tissues and checked for coloration (noted to be yellowish). Immersion of 25 minutes at room temperature in 5 mL fresh medium and incubated for 17 hour and 45 minutes in 1 mL fresh mediu, 37ºC, 5% CO2.

NUMBER OF REPLICATES: 4

NEGATIVE CONTROL USED:
distilled water - ADL Prochillab - Batch No. 211021

POSITIVE CONTROL USED:
Methyl acetate - Sigma Aldriche, batch No. BCBX8836.

APLICATION DOSE AND EXPOSURE TIME:
Positive and negative control dose: 50 μL: applied to the surface of 2 RhCE tissue replicates for 5 hours and 55 minutes.
Test item: 50 mg: applied to the surface of 2 RhCE tissue replicates for 5 hours and 55 minutes.

POST TREATMENT DURATION
17 hour and 45 minutes post exposure incubation

CELL VIABILITY MEASUREMENTS:
Measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.
RhCE constructs were placed in 300 μL of a MTT solution at 1.0 mg/mL (3h and 3 minutes). The precipitated blue formazan product was then extracted form the lower layer of the tissues by placing each insert in 2 mL of isopropanol (1h and 57 minutes).
Concentration of formazan was measured by determining the OD at 570 nm.

OD MEASURES:
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 ELx800 absorbance microplate reader supplied by BioTek and the validated software Gen5 ELISA

EVALUATION AND INTERPRETATION OF RESULTS:

OD values were used to calculate the mean percent tissue viability normalized to the negative control, which was set to 100%. The percentage tissue viability cut-off value distinguishing classified from non-classified test items is 60%. Results interpretation:
- Test item is identified as not requiring classification and labelling according to UN GHS No Category: if mean percent tissue viability after exposure and post-exposur incubation is >60%. In this case, no futher testing in other test methods required.
- The item is identified as potentially requiring classification and labelling accoding to UN GHS (Category 2 or Category 1): if mean percent tissue viability after exposure and post-exposure incubation is ≤60%. futher testing with other methods will be required because the RhCE tets method shows a certain number of false positive and cannot revolve between UN GHS Categories 1 and 2.

Results and discussion

In vitro

Results

Irritation parameter:

percent tissue viability 

Run / experiment:

Mean corrected percent tissue

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Potentially requiring classification and labeling according to UN GHS Category 2 or Category 1.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- The difference of viability between two tissue replicates should be less than 20%
- Acceptance criteria met for negative control: OD values of the two replicates should be in the range > 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.
- Acceptance criteria met for positive control: Tissues trated with the positive control substance should show a mean tissue viability <50%.

The difference between the replicates was higher than 20% for positive control. As the mean tissue viability of positive control is less than 50%, this deviation is considered as without impact on the conclusion of the study.

Any other information on results incl. tables

	Well ID	OD	Mean OD / disc	Mean OD / product	Viability %	Mean viability %	SD viability %	Viability difference between replicates %	Conclusion
Negative Control	SPL1	0.804 0.776 0.810	0.797	0.784	101.7	100.0	2.3	3.3	No Category
	SPL2	0.711 0.826 0.775	0.771		98.3
Positive control	SPL 3	0.514 0.513 0.486	0.504	0.375	64.3	47.8	23.4	33.0	UN GHS Category 2 or 1
	SPL 4	0.237 0.255 0.244	0.245		31.3
Test item PH-ZZ/0351	SPL 9	0.068 0.070 0.073	0.070	0.068	8.9	8.7	0.4	0.5	UN GHS Category 2 or 1
	SPL 10	0.066 0.065 0.066	0.066		8.4
Test item PH-22/0351 NSMTT	SPL 7	0.047 0.047 0.047	0.047	0.047	6.0	5.9	0.1	0.1
	SPL 8	0.046 0.045 0.046	0.046		5.9
Test item PH-22/0351 corrected						2.7

Applicant's summary and conclusion

Interpretation of results:

other: No prediction can be made (CLP Regulation EC no. 1272/2008)

Conclusions:

Under the experimental conditions adopted and inaccordance with the Regulation EC No. 1272/2008, the test item has to be identified as potentially requiring classification and labelling according to UN GHS Category 2.

Executive summary:

An in vitro RhCE study according to OECD 492 was conducted under GLP conditions to assess the irritation potential of the test item. Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Based on the preliminary tests, two tissues (0.6 cm2) were pre-wetted with 20 µL Ca2+ Mg2+Free-DPBS, incubated for 30 min, then dosed topically with 50 mg test item and exposed for 5 hours and 55 minutes at standard culture conditions.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 17 hours and 45 min at 37ºC, 5% CO2. The MTT assay for cell viability evaluation was performed on the tissues, by incubating them for 3 hours and 3 minutes at standard culture conditions. The precipitated formazan crystals were then extracted using isopropanol and quantified spectrophotometrically (OD at 570 nm) in order to determine cell viability.

Concurrent positive and negative controls were run in parallel. All validity criteria were met. The relative mean viability of the tissues treated with the test item was 2.7%. This value is below the threshold for eye irritation potential (≤ 60%). Test items that induce values below the threshold are considered either eye irritant or inducing serious eye damage. Thus, no prediction can be made.