Phenolphthalein

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November 19,2019 - November 21, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

June 18, 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.69 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:

31 July 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phenolphthalein

EC Number:

201-004-7

EC Name:

Phenolphthalein

Cas Number:

77-09-8

Molecular formula:

C20H14O4

IUPAC Name:

3,3-bis(4-hydroxyphenyl)-1,3-dihydro-2-benzofuran-1-one

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: The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg per tissue

Duration of treatment / exposure:

6 hours (± 15 minutes)

Duration of post- treatment incubation (in vitro):

18 hours (± 15 minutes)

Number of animals or in vitro replicates:

3

Details on study design:

- RhCE tissue construct used:
The EpiOcular™ Tissues (OCL-200, OCL-212) was obtained from MatTek In Vitro Life Science Laboratories. Bratislava. Slovakia.

- Doses of test chemical and control substances used :
Solid test item: 50 mg per tissue
Negative control: 50 µL per tissue
Positive control: 50 µL per tissue

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
The tissues were incubated at 37°C and 5% CO2 for 6 hours (± 15 minutes). Post-treatment Incubation was for 18 hours (± 15 minutes) at 37°C and 5% CO2.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
For correct interpretation of results, it is necessary to assess the ability of a test item to directly reduce MTT. For this purpose, a 1.0 mg/mL MTT solution (in DMEM) was prepared. 50 mg of the test item were added to 1 mL of the MTT solution in a 6-well plate and the mixture was incubated at 37 ± 1°C and 5% CO2 and protected from light for 3 hours (± 5 minutes). Sterile deionised water (50 uL) was used as negative control concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.

- Number of tissue replicates used per test chemical and controls: 3

- Description of the method used to quantify MTT formazan :
After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37°C and 5% CO2. The inserts were removed from the 24-well plate after 180 minutes (±10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 6-well plate containing 2 mL isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plate was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically. The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :
The test item is identified as not requiring classification and labeling according to UN GHS (No Category) if the mean percent tissue viability is more than 60%. In this case no further testing in other test methods is required.
If the mean percent tissue viability is less than or equal 60%. no prediction can be made. In this case, further testing with other test methods will be required because PdiCE test methods show a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.

- Acceptance Criteria:
1. The negative control OD is >0.8 and <2.5
2. The mean relative viability of the positive control is:
a) 30-minute exposure (treatment of liquid test items): below 50% of control viability
b) 6-hour exposure (treatment of solid test items): below 50% of control viability
3. The difference of viability between the two relating tissues of a single chemical is <20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Results

Irritation parameter:

other: % viability

Run / experiment:

1st run

Value:

113.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

34.1%

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer.

ACCEPTANCE OF RESULTS:
1. The negative control OD is >0.8 and <2.5 (1.924 and 2.060).
2. The mean relative viability of the positive control is below 50% of the negative control viability (34.1%).
3. The difference of viability between the two relating tissues of a single chemical is <20% (values between 3.7% to 6.8%) in the same run (for positive and negative control tissues and tissues of single chemicals).
The study met all acceptance criteria.

Any other information on results incl. tables

Table 1: Results

Group	Tissue 1		Tissue 2		Mean		SD	Difference between tissue replicates
		OD	Viability	OD	Viability	OD	Viability
Negative Control	1.924	96.6%	2.060	103.4%	1.992	100.0%	4.81	6.8%
Positive Control	0.633	31.8%	0.725	36.4%	0.679	34.1%	3.25	4.6%
Test item	2.217	111.3%	2.290	115.0%	2.254	113.2%	2.62	3.7%

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item did not show an eye irritating potential in the EpiOcular™-model.

Executive summary:

A study according OECD TG 492 was conducted to investigate the potential of the test to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. After treatment with the negative control (sterile deionized water) the OD was 1.924 and 2.060 of the two treated tissue, respectively (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 34.1% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 113.2% and, thus, higher than 60%,i.e.according to OECD 492 the test item is identified as not requiring classification and labeling according to UN GHS (No Category).