Phenyl 4-hydroxybenzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-08 to 2018-03-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 491 (Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted: July, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phenyl 4-hydroxybenzoate

EC Number:

241-698-9

EC Name:

Phenyl 4-hydroxybenzoate

Cas Number:

17696-62-7

Molecular formula:

C13H10O3

IUPAC Name:

phenyl 4-hydroxybenzoate

Specific details on test material used for the study:

- Batch: 018964K19K
- CAS No.: 17696-62-7
- Purity: 99.1%
- Appearance: White solid powder
- Expiry Date: 2018-08-02
- Storage Conditions: At room temperature
- Stability in Solvent: Not indicated by the Sponsor
- Purpose of Use: Industrial chemical

Test animals / tissue source

Species:

rabbit

Details on test animals or tissues and environmental conditions:

The rabbit corneal cell line SIRC was used for performing the STE test method. SIRCs are growing as confluent monolayers.

Test system

Vehicle:

other: Mineral oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 200 µL
- Concentration (if solution): 0.5 % (v/v), 0.05% (v/v)

Duration of treatment / exposure:

5 minutes

Observation period (in vivo):

n.a.

Duration of post- treatment incubation (in vitro):

After exposure, cells were washed twice with 0.2 mL of PBS and 0.2 mL MTT solution (0.5 mg MTT/mL of MEM) was added. After a two-hour reaction time at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere the MTT solution was decanted. MTT formazan was extracted by adding 0.2 mL of 0.04 N hydrochloric acid – isopropanol for approximately 60 minutes in the dark at room temperature and the absorbance of MTT formazan solution was measured with a microplate reader.

Number of animals or in vitro replicates:

triplicates per treatment group, three independent runs

Details on study design:

CELL CULTURE:
Large stocks of the SIRC cell line (supplied by ATCC) were stored in liquid nitrogen in the cell bank of Envigo CRS GmbH allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 ± 1.5 °C in plastic flasks (e.g. NUNC) containing a culture medium comprising Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 units/mL penicillin and 100 µg/mL streptomycin..
The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere. Cells were propagated 2 to 3 passages in a culture flask before being employed for testing and did not exceed 25 passages from thawing

SEEDING THE CULTURES:
Exponentially growing stock cultures more than 50% confluent were rinsed with PBS and treated with Trypsin at 37 ± 1.5 °C for 5 minutes (Gibco BRL Trypsin/EDTA Solution 10x Kat. Nr. 35400-019). Then the enzymatic digestion was stopped by adding complete medium and a single cell suspension was prepared.
Individual wells of a 96-well tissue-culture microtiter plate were inoculated with 0.2 mL complete medium containing approximately 3 x 104 cells/mL in case that the cells were seeded four days prior to the treatment and 1.5 x 104 cells/mL in case that the cells were seeded 5 days prior to the treatment. The plates were incubated at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere for 4 days and 5 days, respectively. Cells should reached a confluence of more than 80% at the time of testing.

TREATMENT:
The test item was tested in three independent runs (with different cell cultures and on different days).
On the day of the experiments right before application, the test item was stably suspended in mineral oil to reach a final concentration of 5% (w/w). Following, this solution was diluted by serial 10-fold dilution with the respective solvent to reach final concentrations of 0.5% (v/v) and 0.05% (v/v).
The test item was prepared freshly prior to each experiment.
For the treatment the complete medium was removed and the cells were re-fed with 200 µL treatment solution containing negative, solvent and positive control as well as the two different concentrations of the test item (5% and 0.05%) and the medium blank, respectively. All dose groups were tested 3 times.
The exposure period was 5 minutes at room temperature


CELL VIABILITY MEASUREMENT:
After exposure, cells were washed twice with 0.2 mL of PBS and 0.2 mL MTT solution (0.5 mg MTT/mL of MEM) was added. After a two-hour reaction time at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere the MTT solution was decanted. MTT formazan was extracted by adding 0.2 mL of 0.04 N hydrochloric acid – isopropanol for approximately 60 minutes in the dark at room temperature and the absorbance of MTT formazan solution was measured with a microplate reader (Versamax® Molecular Devices) at 570 nm (without a reference).

DATA EVALUATION:
The optical density (OD) value obtained from the test item was used to calculate cell viability relative to the solvent control, which is set at 100%. The relative cell viability is expressed as a percentage and obtained by dividing the OD of the test item by the OD of the solvent control after subtracting the OD of blank from both values. Similarly, the relative cell viability of each solvent control is expressed as a percentage and obtained by dividing the OD of each solvent control by the OD of the medium control after
subtracting the OD of blank from both values. The arithmetic mean of the three wells of the test item and solvent control in each independent repetition was used to calculate the arithmetic mean of relative cell viability. The final arithmetic mean of the cell viability was calculated from the three independent runs.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Mean cell viability (%)

Remarks:

0.05 %

Run / experiment:

Mean of three replicates

Value:

71.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Results 2

Irritation parameter:

other: Mean cell viability (%)

Remarks:

5 %

Run / experiment:

Mean of three replicates

Value:

58.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Table 2: Summary of Results

Test Group	Cell Viability [%] per Test			Mean Cell Viability [%]	Standard Deviation [%]	Predicted Eye Irritation Potential
	Test 1	Test 2	Test 3
Medium Control	84.7	98.0	97.3	93.3	7.5
Solvent Control (0.9% NaCl)	100.0	100.0	100.0	100.0	-
Solvent Control (mineral oil)	100.0	100.0	100.0	100.0	-
Positive Control	13.2	19.1	34.5	22.3	11.0
Test Item 0.05%	73.8	60.0	80.3	71.4	10.4	No prediction can be made
Test Item 5%	68.2	55.4	51.9	58.5	8.6
Solvent control (0.9% NaCl)compared to medium control	118.1	102.1	102.8
Solvent control (mineral oil) compared to medium control	143.2	115.2	113.0

Applicant's summary and conclusion

Interpretation of results:

other: no prediction can be made

Conclusions:

In this study, under the given conditions, no prediction can be made regarding the eye irritation potential of Phenyl 4-hydroxybenzoate.

Executive summary:

In the present study the eye irritation potential of Phenyl 4-hydroxybenzoate (purity 99.1 %) was analysed using the Short Time Exposure in vitro test method for identifying eye irritation according to OECD 491. The test item was diluted with mineral oil to give a 5 and 0.05 % concentration. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 5-min exposure and compared to those of the concurrent negative controls. Toxic effects were observed following incubation with the highest tested test item concentration of 5% in all three independent tests. The cell viabilities were reduced below 70% (viability range between 51.9% and 68.2%). For the 0.05% test item concentration only the second test showed a cell viability below 70% (60.0%). However, the mean cell viability of the 0.05% test item concentration treated cells did not drop below 70% cell viability (viability range between 60.0% and 80.3%), therefore a prediction of the eye irritation potential of Phenyl 4-hydroxybenzoate according to the UN GHS classification is not possible.