Registration Dossier

Administrative data

Description of key information

The potential of Phenyl 4-hydroxybenzoate to induce skin irritation/corrosion was evaluated in two suitable in vitro test methods (OECD 439 and OECD 431). Based on the results, Phenyl 4-hydroxybenzoate (99.1% purity) must be considered as irritant to skin and is therefore classified as Skin Irrit. 2, H315 in accordance with the CLP criteria.

The eye irritation potential of the target substance was investigated in two suitable in vitro test methods (OECD 491, OECD 438). By assessing the results from both in vitro eye irritation tests, the substance must be considered a non-irritant to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-02-20 to 2018-02-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted: 28th July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Sponsor’s identification: PHENYL 4-HYDROXYBENZOATE
- Batch No.: 018964K19K
- CAS No.:17696-62-7
- Storage: room temperature
- Form: powder
- Colour: white
- Expiry date: 2018-10-01
- Purity: 99.1%
Test system:
human skin model
Source species:
human
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The 0.6 cm² reconstituted epidermis (epiCS, Cell Systems – Batch No. 100-AH0252-1) were received on 20 February 2018. The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The insert was placed in a 6 wells culture plate which had been previously filled with 1 mL of culture medium (Cell Systems Batch No. 305-AH-0432). The culture dishes were incubated at 37 ± 2 °C, 5% CO2 during 21 hours and 23 minutes before treatment. Just before the treatment, the culture medium was replaced by a new culture medium (Cell Systems, Batch No. 305-AH-0432).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 min), 37 °C ± 1 °C, 5% ± 1% CO2 (1 hour)
- Temperature of post-treatment incubation (MTT incubation): 37 ± 1 °C (for 3 h, 5.0% CO2)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 3 minutes and 1 hour after the test item application the human epidermis were washed 20 times with 1 mL of DPBS (DPBS – Dutscher, Batch No. 5691217).
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h (at 37 ± 1 °C)
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: max. ± 30 nm

PREDICTION MODEL / DECISION CRITERIA:
See Table 1 in box ‘Any other information on materials and methods incl. tables’
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL KOH
- Concentration (if solution): 8N

Negative CONTROL
- Amount(s) applied (volume or weight): 50 µL distilled water
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The test item was applied as supplied at the dose of 25 mg to the epidermal surface of the 2 living human skin models previously moistened with 25 μL of distilled water, during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8N
Duration of treatment / exposure:
3 minutes and 1 hour
Duration of post-treatment incubation (if applicable):
3 h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 min exposure
Run / experiment:
Mean of two replicates
Value:
100.51
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
60 min exposure
Run / experiment:
Mean of two replicates
Value:
99.87
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:
- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: no
- Colour interference with MTT: no


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

For individual results see Table 2 in box "Any other information on results incl. tables".

Table 2: Results of main experiment

 

Mean viability (%)

3 min exposure

1 hour exposure

Conclusion

Positive control

6.08

0.39

Category 1A

Test item

100.51

99.87

Non Corrosive

Interpretation of results:
other: non-corrosive
Conclusions:
In this study under the given conditions the test item showed no corrosive effects. The test item is therefore not classified as “non-corrosive“ in accordance with UN GHS Criteria "Category 1".
Executive summary:

In a primary skin corrosion study conducted according to guideline OECD 431, two EpiDerm tissues per dose group were exposed to 25 mg of Phenyl 4-hydroxybenzoate, chloride (99.1 % purity) for 60 min and 3 min each and cytotoxicity was measured in comparison to the concurrent negative controls. Irritation was scored by the method of mean relative tissue viability. No corrosive effects were observed after treatment with the test item. The mean relative tissue viability (% negative control) of the epidermis skins treated with the test item were 100.51% (considered as 100%) and 99.87%. In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified for Category 1 “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-12 to 2018-02-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted: 28th July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Sponsor’s identification: PHENYL 4-HYDROXYBENZOATE
- Batch No.: 018964K19K
- CAS No.: 17696-62-7
- Storage: room temperature
- Quantity: 38.90 g (container + content)
- Form: powder
- Colour: white
- Expiry date: 2018-10-01
- Purity: 99.1% of phenyl 4-hydroxybenzoate
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The 0.50 cm² reconstructed epidermises (Episkin SA, RHE/S/17 Batch No. 18-RHE-002) were received on 16 January 2018. The same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 1 mL of growth medium (Episkin SA, batch No. 18 SGM 003) during 2 hours and 25 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 μL of maintenance medium (Episkin SA, batch No. 18 SMM 002).
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (exposure duration: 42 min)
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C (5.0% CO2 for 42 h ± 5 min) in fresh medium

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 42 minutes after the test item application, the nylon mesh was removed and the human epidermises were washed with 25 x 1 mL of DPBS (Dutscher - Batch No. 3941117). The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 h ± 5 min
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
The test substance is considered to be not irritating if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is >50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
PREPARATION AND APPLICATION OF THE TEST ITEM
The test item was applied, as supplied, during 42 minutes at room temperature at the dose of 16 mg to the epidermal surface of 3 living Reconstructed Human epidermis.
NEGATIVE CONTROL: DPBS
POSITIVE CONTROL: 5% sodium dodecyl sulfate
TEST ITEM: 16 mg
Duration of treatment / exposure:
42 min
Duration of post-treatment incubation (if applicable):
42 h +/- 5 min
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of three tissues
Value:
31.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

For individual results see Table 1 in box "Any other information on results incl. tables".

Table 1: Results of the main experiment

Test group

Mean OD

Mean viability (%)

Conclusion

Negative control

1.002

100.0

 

Positive control

0.010

1.0

Irritant

Test item

0.320

31.9

Corrosive or Irritant

Interpretation of results:
other: Category 1 or Category 2
Conclusions:
In conclusion, in accordance with the Regulation EC No. 1272/2008 and in absence of information on skin corrosion, the test item has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”. Further testing is required.
Executive summary:

In primary dermal irritation study conducted according to OECD Guideline 439 (Reconstructed Human Epidermis Test), the EpiDerm Episkin SA, RHE/S/17, was topically exposed to Phenyl 4-hydroxybenzoate (99.1 % purity) for 42 min followed by a 42 h post-incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability (% negative control) was ≤ 50% (31.9%) after 60 min treatment and 42 h post-incubation. Based on this result, Phenyl 4-hydroxybenzoate is considered to be corrosive or irritating to the skin in accordance with UN GHS "Category 1 or 2" and further testing is required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
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. certificate)
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
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.
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.
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
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

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

 

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.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-08-20 to 2018-10-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Purity: 99.1%

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Source: test material was provided by the Sponsor; batch no.: 018964K19K
- Expiration date of the lot/batch: 02 October 2018
- Purity test date: 02 August 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the purpose of this study the test item was used as supplied.
Species:
chicken
Strain:
other: Gallus gallus
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES

- Source: Baileys Turkeys Ltd., Cheshire, UK
- Number of animals: Multiple chicken heads were used.
- Characteristics of donor animals (e.g. age, sex, weight): The chickens weighed approximately 3 kg and were approximately 56 days old prior to being humanely killed for human consumption.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
- Time interval prior to initiating testing: Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline.
- Indication of any existing defects or lesions in ocular tissue samples: Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds.
- Indication of any antibiotics used: not specified
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): 0.03 g
- Concentration (if solution): The test item was applied undiluted.
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES: Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are ≤ 0.5.
Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt-tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.
Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ± 1.5 °C.
Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea.
Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.
After the approval process the eyes were incubated for approximately 45 minutes for equilibrium purposes.

EQUILIBRATION AND BASELINE RECORDINGS: Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES: 3 for test item and positive control; 2 for the negative control

NEGATIVE CONTROL USED: Sodium chloride 0.9% w/v

SOLVENT CONTROL USED (if applicable): None; the test item was applied undiluted.

POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 0.03 g of test item was applied to the cornea. The entire surface of the cornea was evenly covered. The test item remained in place for 10 seconds.

OBSERVATION PERIOD: 30, 75, 120, 180 and 240 minutes after exposure

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL of isotonic saline

METHODS FOR MEASURED ENDPOINTS:
Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test substance exposure) were determined at each of the above time points.
After the final examinations at the 240-minute time point, the eyes were preserved in neutral buffered formalin for possible histopathology. Eyes were retained until finalization of the final report. Percentage corneal swelling was assessed from corneal thickness measurements. The calculation was expressed in the following formula:
((corneal thickness at time (t) – corneal thickness at time = 0)/(corneal thickness at time = 0)) x 100
Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.
Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
The mean fluorescein retention scores for all test eyes are calculated at the 30-minute time interval only. These measurements are used for the overall classification for each test item.
Pitting, sloughing, roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.

SCORING SYSTEM:
- Mean maximum opacity score
Score Description of score
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent area; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil barely discernible
4 Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment
Score Description of score
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA: Decision criteria as indicated in the OECD test guideline was used (see Table 1 in box "Any other information on materials & methods incl. tables).
Irritation parameter:
cornea opacity score
Run / experiment:
Test item - Maximal mean score for corneal opacity
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II
Irritation parameter:
fluorescein retention score
Run / experiment:
Test item - Mean score of fluorescein retention
Value:
0.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
percent corneal swelling
Run / experiment:
Test item - Maximal mean corneal swelling compared to time zero
Value:
6.07
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II
Other effects / acceptance of results:
OTHER EFFECTS:
‘Very faint opacity’ was noted in the negative control treated eyes.
‘Complete corneal opacity; iris invisible’ was noted in all positive control treated eyes.
‘Scattered or diffused areas; details of the iris are clearly visible’ (of opacity) were noted in the test item treated eyes.
No morphological effects were noted in the negative control item and positive control item treated eyes. Test item adherence was noted in all test item treated eyes.
‘No fluorescein retention’ was noted in the negative control treated eyes during the study period.
‘Confluent large areas of the cornea retaining fluorescein’ was noted in all positive control treated eyes.
‘Very minor single cell staining’ was noted in the test item treated eyes.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: No

Table 2: Individual Scores and Mean Scores for Corneal Effects - Test item

Endpoint Eye Number Time (after decontamination)
0 minutes 30 minutes  75 minutes 120 minutes 180 minutes 240 minutes
Corneal Opacity 3A 0.5 0.5 0.5 1 1 1
6A 0.5 0.5 1 1 1 1
8A 0.5 0.5 0.5 0.5 1 1
Mean 0.5 0.5 0.7 0.8 1.0 1.0
ICE Class II
Fluorescein Retention 3A - 0.5 - - - -
6A - 0.0 - - - -
8A - 0.5 - - - -
Mean - 0.3 - - - -
ICE Class I
Corneal Thickness 3A 0.72 0.74 0.72 0.74 0.78 0.80
6A 0.72 0.78 0.77 0.73 0.74 0.76
8A 0.70 0.75 0.73 0.73 0.70 0.70
Mean 0.71 0.76 0.74 0.73 0.74 0.75
Mean Corneal Swelling (%) - 6.07 3.74 2.80 3.74 5.61
ICE Class II
Epithelium Condition 3A - TA TA TA TA TA
6A - TA TA TA TA TA
8A - TA TA TA TA TA
Mean - TA TA TA TA TA
ICE Classes Combined 1 x I, 2 x II

TA = Test Item Adherence

Table 3: Individual Scores and Mean Scores for Corneal Effects - Positive control

Endpoint Eye Number Time (after decontamination)
0 minutes 30 minutes  75 minutes 120 minutes 180 minutes 240 minutes
Corneal Opacity 2A 0 4 4 4 4 4
5A 0 4 4 4 4 4
7A 0.5 4 4 4 4 4
Mean 0.2 4.0 4.0 4.0 4.0 4.0
ICE Class IV
Fluorescein Retention 2A - 3 - - - -
5A - 3 - - - -
7A - 3 - - - -
Mean - 3.0 - - - -
ICE Class IV
Corneal Thickness 2A 0.74 0.80 0.92 0.82 0.90 0.96
5A 0.74 0.80 0.82 0.92 0.90 0.88
7A 0.70 0.84 0.84 0.85 0.90 0.92
Mean 0.73 0.81 0.86 0.86 0.90 0.92
Mean Corneal Swelling (%) - 11.93 18.35 18.81 23.85 26.61
ICE Class III
Epithelium Condition 2A - N N N N N
5A - N N N N N
7A - N N N N N
Mean - N N N N N
ICE Classes Combined 1 x III, 2 x IV

N = normal

Table 4: Individual Scores and Mean Scores for Corneal Effects - Negative control

Endpoint Eye Number Time (after decontamination)
0 minutes 30 minutes  75 minutes 120 minutes 180 minutes 240 minutes
Corneal Opacity 1A 0 0 0 0.5 0.5 0.5
4A 0.5 0.5 0.5 0.5 0.5 0.5
Mean 0.3 0.3 0.3 0.5 0.5 0.5
ICE Class I
Fluorescein Retention 1A - 0 - - - -
4A - 0 - - - -
Mean - 0.0 - - - -
ICE Class I
Corneal Thickness 1A 0.73 0.72 0.74 0.70 0.70 0.70
4A 0.73 0.73 0.74 0.70 0.72 0.72
Mean 0.73 0.73 0.74 0.70 0.71 0.71
Mean Corneal Swelling (%) - -0.68 1.37 -4.11 -2.74 -2.74
ICE Class I
Epithelium Condition 1A - N N N N N
4A - N N N N N
ICE Classes Combined 3 x I

N = normal

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed no corrosive effects. The test item does not require classification for eye irritation or serious eye damage in accordance with UN GHS “No Category” for eye irritation.
Executive summary:

In a primary eye irritation study conducted according to OECD 438 (Isolated Chicken Eye Test), 0.03 g of the test item phenyl 4-hydroxybenzoate (purity: 99.1%) was applied onto the cornea of three enucleated eyes of Gallus gallus (Spring chickens). Three other enucleated eyes were treated with positive control (imidazole) and two other enucleated eyes were treated with negative control (sodium chloride 0.9% w/v). The test item or the respective controls remained in place for 10 seconds and were removed from the eyes afterwards by rinsing with 20 mL of isotonic saline. Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (± 5 minutes) after the eyes had been decontaminated with the isotonic saline.

Scattered or diffused areas; details of the iris are clearly visible (of opacity) were noted in the test item treated eyes. No morphological effects were noted in the negative control item and positive control item treated eyes. Test item adherence was noted in all test item treated eyes. Very minor single cell staining was also noted in the test item treated eyes.

The ocular reactions observed in test item treated eyes were as follows:

Maximal mean score for corneal opacity: 1.0; ICE Class II

Mean score of fluorescein retention: 0.3; ICE Class I

Maximal mean corneal swelling compared to time zero: 6.07%; ICE Class II

 

In this study under the given conditions the test item showed no corrosive effects. The test item does not require classification for eye irritation or serious eye damage for eye irritation in accordance with CLP criteria.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The potential of Phenyl 4-hydroxybenzoate to induce skin irritation/corrosion was evaluated in two suitable in vitro test methods (OECD 439 and OECD 431). In the study conducted according to OECD 439, the mean relative tissue viability (% negative control) was ≤ 50% (31.9%) after 60 min treatment and 42 h post-incubation. Based on this result, Phenyl 4-hydroxybenzoate is considered to be corrosive or irritating to the skin in accordance with UN GHS "Category 1 or 2". Further, a study was conducted according to OECD 431, which distinguishes between corrosive and non-corrosive substances. In this study, no corrosive effects were observed after treatment with the test item. Considering the results from both studies in a weight-of-evidence approach, it can be concluded that the substance is considered to be an irritant to the skin and does warrant classification as Skin Irrit. 2, H315.

The eye irritation potential of the target substance was investigated in the Short Time Exposure in vitro test method for identifying eye irritation conducted according to OECD 491. In this study, it was shown that the substance is neither corrosive nor non-classified and no prediction can be made regarding the eye irritation potential of Phenyl 4-hydroxybenzoate. Thus, a further study according to OECD 438 was conducted. In this study, the substance showed no corrosive effects. Based on the results from this study, the substance is not considered to be irritant to the eye and thus, no classification for eye irritation is warranted.

Justification for classification or non-classification

Based on the results from two in vitro skin irritation/corrosion tests (OECD 431/439), classification as Skin Irrit. 2, H315 is warranted. Based on the results from the in vitro eye irritation studies conducted according to OECD 491 and OECD 438, the test substance is not considered to be an eye irritant and thus, no classification for eye irritation is warranted.