Registration Dossier

Administrative data

Description of key information

The potential of Ethoxylated phenol phosphate (100% purity) to induce skin irritation (OECD 431, 439) and eye irritation (OECD 437, OECD 438) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered non-irritant to the skin. By assessing the results from both in vitro eye irritation tests in a weight-of-evidence approach, the target substance must be considered as irritating to the eye (Eye Irrit. 2, H319)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion, other
Remarks:
Skin corrosivity and irritation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-10 to 2018-03-26
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- CAS number: 39464-70-5
- Appearance: Clear, yellow-amber liquid
- Purity: 100%
- Storage conditions: Room temperature (15-25 °C, ≤ 70 % relative humidity (RH))
- Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The corrosivity and irritation potential of a chemical may be predicted by measurement of its cytotoxic effect, as reflected in the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] assay, on EPISKIN^TM(SM) reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD No. 404) and is specifically approved as a replacement for the in vivo skin corrosivity and irritation tests within OECD No. 431. and OECD No. 439.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN (SM)
- Tissue batch number(s): 18-EKIN-002
- Expiry date: 15 January 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg / mL
- Incubation time: 3 h + / - 5 min
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: two for the corrosion test and three for the irritation test

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- N. of replicates : Two negative controls and two positive controls were run in corrosivity testing and three negative controls and three positive controls were run in irritation testing. As the test item was coloured, two additional test item-treated living tissues were used for the non specific OD evaluation.

PREDICTION MODEL / DECISION CRITERIA
OECD 431:
The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).
For 2 disks:
If both disks have mean viability of ≥ 35% = Non Corrosive
If both disks have mean viability of < 35% = Corrosive (at the corresponding incubation period)

For more than 2 disks:
If the mean value is ≥35% and the variability is less than 50% = Non Corrosive
If the mean value is <35% and the variability is less than 50% = Corrosive

Otherwise:
If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35%. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.

OECD 439:
In the present study, the irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item. The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is less or equal (≤) to 50% of the mean viability of the negative controls. In case the test item is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test item is considered to be irritant to skin in accordance with UN GHS Category 2. The test item may be considered to be non-irritant to skin in accordance with UN GHS (No Category), if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is more than (˃) to 50% of the mean viability of the negative controls.

Validity of the Test System:
General validity criteria:
- The mean OD value of the two or three negative control tissues should be ≥ 0.6 and ≤ 1.5
- Control OD values should be within the historical control range.
- The mean OD value of the blank samples (acidified isopropanol) should be < 0.1.
- Specific criteria for corrosivity testing:
The difference of viability between the two tissue replicates should not exceed 30%. The acceptable mean percentage viability range for positive controls is ≤ 20%.

Specific criteria for irritation testing:
Standard deviation value (SD) of the negative control tissues and the identically treated replicates % viability should be ≤ 18. The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be ≤ 18.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
Irritation test
- Amount(s) applied (volume or weight with unit): 20 µL
Corrosion test
- Amount(s) applied (volume or weight with unit): 50 µL

NEGATIVE CONTROL
Irritation test
- Amount(s) applied (volume or weight): 50 µL PBS
Corrosion test
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 0.9% (w/v) NaCl solution))

POSITIVE CONTROL
Irritation test
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 5% (w/v) SDS solution
Corrosion test
- Amount(s) applied (volume or weight): 50 µL glacial acetic acid
Duration of treatment / exposure:
Irritation test: 15 min
Corrosion test: 4 h
Duration of post-treatment incubation (if applicable):
42 h for irritation testing
Number of replicates:
- Two for corrosion test and two for irritation test
- Two negative controls and two positive controls were run in corrosivity testing and three negative controls and three positive controls were run in irritation testing . As the test item was coloured, two additional test item-treated living tissues were used for the non specific OD evaluation.
Irritation / corrosion parameter:
% tissue viability
Remarks:
OECD 431
Run / experiment:
mean of replicates
Value:
82.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
OECD 439
Run / experiment:
mean of triplicates
Value:
97.2
Vehicle controls validity:
not examined
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: None

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions, the test item showed no irritant or corrosive effects. The relative mean tissue viability was > 50% for irritation and > 35 % for corrosivity testing. The test item is therefore classified as “non-irritant” and "non-corrosive" in accordance with UN GHS “No Category”.
Executive summary:

In a combined in vitro skin irritation/corrosion study conducted according to OECD guidelines 431 and 439, the skin corrosion and irritation potential of the test item, Ethoxylated phenol phosphate, was analysed by using the three-dimensional human skin model EpiSkin-SM (SkinEthic) comprising a reconstructed human epidermis with functional stratum corneum. The mean cell viability was above the threshold of 35% (82.4%) in the corrosivity test and above the threshold of 50% (97.2%) in the irritation test, as compared to the negative control. Based on these results, the test item is considered to be non - irritant and non – corrosive to the skin and no classification is warranted.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
2018-03-23 to 2018-04-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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 09 October 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- CAS No.: 39464-70-5
- Description: Clear yellow to amber viscous liquid
- Storage condition: At room temperature
- Purity: 100% (UVCB)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
As the test item was a surfactant containing mixture, it was tested undiluted (i.e. in its original form). Thus, no vehicle was used.
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
- Age of donor animals: up to 12 months old (typically, 5 to 8 months old).
- Transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): the eyes were immerged in containers filled with cooled buffered Hanks medium and placed into a cooling-box with a sufficient amount of ice packs to ensure cooling until arrival at Citoxlab France. Containers with smooth internal surfaces were used for the transport to avoid damage to the corneas. Hank’s medium contained an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]
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): 750 µL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
10 minutes (± 30 seconds)
Duration of post- treatment incubation (in vitro):
2 hours (± 10 minutes)
Number of animals or in vitro replicates:
three
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
The corneas were then used immediately.
Storage of the corneas: as the corneas were not used immediately, they were stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4 °C, for a maximum of 24 hours before use. On the day of use, the corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded.

STUDY DESIGN:
A single experiment was performed. The treatment time was dependent upon the nature of the test item.
Treatment time:
As the test item was a surfactant containing mixture, a treatment time of 10 minutes (± 30 seconds) was used in the study.
Assembly of the corneas and the holders:
The corneas were mounted in the corneal holders with the endothelial side against the O-ring of the posterior chamber. Each cornea was identified with the corresponding holder number.
Pre-incubation:
For pre-incubation, both chambers of the corneal holder were filled to overflowing with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) at room temperature. The posterior chamber was always filled first to maintain the natural concave shape of the cornea.
After making sure that no air bubbles were present within the holder, it was immersed in a water bath, horizontally (cornea positioned vertically), up to approximately three quarters of its height. The holders were pre incubated for 1 hour and 5 minutes (± 5 minutes) at +32 °C (± 1 °C).
At the end of the pre-incubation period, the medium was removed from both chambers of the holder using a metal gavage tube attached to a vacuum pump to ensure complete evacuation. They were refilled with fresh cMEM without phenol red (previously heated to +32 °C), starting with the posterior chamber and taking care that no air bubbles were present. The chambers were re-sealed and the corneas were examined macroscopically through the holder to detect the presence of any defects. Then, the opacity of the cornea was measured to obtain OPT0. Corneas that showed any macroscopic defect or an OPT0 value over 7 were discarded.
Allocation of the corneas:
The test item, the negative and positive controls were tested on three corneas each.

The corneas were distributed as follows:
- the median value of the OPT0 values of all pre-incubated corneas (with OPT0 ≤ 7) was calculated,
- three corneas with opacity values close to the median value were selected as negative control corneas,
- the remaining corneas were shared out between test item and positive control-treated series using a manual distribution procedure.

To rigorously respect the treatment and any other incubation times, the three corneas from the same series were always processed in the same order at each step.
Treatment of corneas:
The medium of the anterior chamber was removed and each item was applied onto the epithelium of the cornea, as specified in the Test item, positive and negative controls application.

The treatment time of each series of three corneas was carefully measured with a chronometer, starting from the beginning of treatment of the first cornea of each series. Then each further operation (rinsing, measurement, etc.) was carried out in the same order for the three corneas of each series. After application of the items, the holders were incubated vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at +32 °C (± 1 °C), for the selected treatment time.
Rinsing of the corneas:
The purpose of rinsing was to eliminate as much items as possible, while taking care not to damage the cornea. On completion of the treatment period, the test item was removed from the front opening of the anterior chamber (open-chamber method) and the epithelium was rinsed as follows:
- any test item adhering to the walls of the anterior chamber was removed using a pipette of heated cMEM (32 °C),
- the corneas were rinsed four times with pre-warmed cMEM containing phenol red (i.e. until the test item had been completely removed from the chamber or until the phenol red was not discoloured). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.

The rinsing efficiency was visually confirmed by observing the transparency and the colour changing of the rinsing medium (containing phenol red). No difficulties were encountered during the rinsing. The anterior chamber was refilled with fresh pre-warmed cMEM without phenol red. The front cover was replaced. Care was taken to make sure that no air bubbles were present within the holders (by ensuring that each chamber was filled to overflowing with pre-warmed cMEM). Following the 10-minute treatment and the rinsing step, the holders were incubated horizontally (corneas placed vertically) for 2 hours (± 10 minutes) in a water bath at +32 °C (± 1 °C). On completion of the 2-hour incubation period, the medium of both anterior and posterior chambers was renewed with pre-warmed cMEM (+32 °C (± 1 °C)), the second opacity measurement (OPT2) was then performed.

Opacity measurements:
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded. Corneal opacity was determined by reading each holder in the right-hand chamber of the calibrated opacitometer, versus an empty holder (without cMEM, cornea and glasses), placed in the left-hand chamber. For opacity measurement, care was taken to make sure that no air bubbles were present within the holders containing corneas (by ensuring that each compartment was filled to overflowing with heated cMEM) and each holder was wiped dry.
Permeability determination:
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution. As the test item was a mixture containing surfactants, the concentration of the fluorescein solution was 4 mg/mL. Before use, the fluorescein solution was validated. For this purpose, the solution of fluorescein was diluted in cMEM in order to obtain a 5 µg/mL solution and the Optical Density at a wavelength of 490 nm (OD490 nm) of this dilution was measured. As the value obtained was between 0.850 and 0.940 the fluorescein solution was validated. For each series of three corneas, a chronometer started from the fluorescein solution application time of the first cornea of the series. The holders were incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at +32 °C (± 1 °C) for 90 minutes (± 5 minutes). At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube. The medium was homogenized prior to determination of OD490 nm, using single-use cuvettes (1 cm path length) and a spectrophotometer (cMEM used as the blank).
Macroscopic examination:
After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium. Then, the corneas were discarded.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of triplicates
Value:
46
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
no prediction can be made
Other effects / acceptance of results:
Macroscopic examination:
Fluorescein fixation was observed on one cornea treated with the negative control. Opacity, fluorescein fixation and thickening of the corneas were observed on the corneas treated with the test item and positive control.

In vitro Irritancy Score:
The individual and mean opacity and permeability values for the test item, positive control and negative control were measured and all acceptance criteria were fulfilled. The study was therefore considered as valid. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was 46. As the mean IVIS was > 3 and ≤ 55, the eye hazard potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category). For detailed results please refer to Table 1 in box "Any other information on results incl. tables".

Table 1: Results for the test item

 

Holder

OPT0

OPT2

OPT2-OPT0

Neg correction

cOPT

OD490nm

Neg correction

cOD490nm

IVIS

Test item

1

-4

19

23

23

22

1.532

1.532

1.525

45

2

-3

12

15

15

14

1.444

1.444

1.437

36

3

-3

20

23

23

22

2.380

2.380

2.373

58

Mean

 

 

 

 

19.3

 

 

1.778

46

S.D.

 

 

 

 

4.6

 

 

0.517

11.1

 

Interpretation of results:
other: no prediction can be made
Conclusions:
In conclusion, based on the mean in vitro irritation score of 46 obtained in the bovine corneal opacity and permeability assay (OECD 437), no prediction can be made regarding the classification of the test substance.
Executive summary:

The eye irritation potential of Ethoxylated phenol phosphate (100% purity) was investigated in the bovine corneal opacity and permeability assay (OECD 437). A mean in vitro irritation score of 46 was determined. The positive control induced the appropriate responses, indicating the validity of the assay. According to the UN GHS criteria, this mean in vitro irritation score does not allow to make any prediction regarding classification of the test item.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-08 to 2018-03-14
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 Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted 9 October 2017
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- CAS number: 39464-70-5 - Appearance: Clear, yellow-amber liquid
- Purity: 100%
- Storage conditions: Room temperature (15-25 °C, ≤ 70 % relative humidity (RH)).
- Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was applied as supplied, no formulation was required.
Species:
chicken
Strain:
other: COBB 500
Details on test animals or tissues and environmental conditions:
- Justification of the test method: The Enucleated Eye Test with isolated eyes of chickens has been recognized as a valuable alternative to the Draize eye irritation test regarding ocular corrosivity or severe eye irritancy testing, because it represents a test system nearest to the in vivo test, without the need to use live animals. In the Isolated Chicken Eye Test (ICET) the test compound is applied in a single dose onto the cornea of isolated eyes, which are obtained from slaughter animals.
- Description of the cell system used: Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to Citoxlab Hungary Ltd. at ambient temperature at the earliest convenience. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Citoxlab Hungary Ltd. and processed within 2 hours of collection.
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): 30 µL
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Number of animals or in vitro replicates:
Three test item treated eyes, three positive control treated eyes and one negative control treated eye
Details on study design:
SELECTION AND PREPARATION OF CORNEAS + QUALITY CHECK OF THE ISOLATED CORNEAS
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity. The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

NUMBER OF REPLICATES: test item & positive control: three eyes; negative control: one eye

NEGATIVE CONTROL USED:
- Name: Physiological saline (Salsol solution 0.9 % (w/v))
- Lot number: 72522Y05-2
- Manufacturer: B. Braun Pharmaceuticals SA
- Expiry date: 31 May 2020
- Storage condition: Room temperature

POSITIVE CONTROL:
- Name: Benzalkonium chloride solution, 50 % in water
- CAS Number: 63449-41-2
- Manufacturer: Sigma-Aldrich Co.
- Batch number: SZBC2960V
- Expiry date: 25 March 2018
- Storage conditions: Room temperature

TREATMENT METHOD
After the zero reference measurements, the eye (in its retainer) was removed from the chamber, held in a horizontal position and the test item was applied onto the centre of the cornea, taking care not to damage or touch the cornea. ~30 µL* of test item was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance. Negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution); positive control eyes were treated with 30 µL 5% (w/v) Benzalkonium chloride solution. One eye was treated with physiological saline, three eyes with the test item and another three eyes with 5% (w/v) Benzalkonium chloride solution.

TEST ITEM REMOVAL
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible. Additional gentle rinsing with 20 mL saline was performed at each time point when the test item on the cornea was observed. The test item treated eyes were rinsed additional gentle rinsing with 20 mL saline after treatment.

OBSERVATION AND ASSESSMENT
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable. Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements. Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.

MEASUREMENT OF ENDPOINTS
Corneal swelling, corneal opacity and fluorescein retention were calculated. The mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change were calculated.
Irritation parameter:
cornea opacity score
Run / experiment:
mean of triplicates
Value:
2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class III
Irritation parameter:
percent corneal swelling
Remarks:
at up to 75 min
Run / experiment:
mean of triplicates
Value:
2.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class I
Irritation parameter:
percent corneal swelling
Remarks:
at up to 240 min
Run / experiment:
mean of triplicates
Value:
6.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class II
Irritation parameter:
fluorescein retention score
Run / experiment:
mean of triplicates
Value:
3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class IV
Other effects / acceptance of results:
Based on this in vitro eye irritation in the isolated chicken eyes test with Ethoxylated phenol phosphate, the test item is not classified as a severe irritant and not classified as non-irritant. For detailed results please refer to Table 5 in box "Any other information on results incl. tables".

OTHER EFFECTS:
- Visible damage on test system: None

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

Table 5: Results on the test item

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

2.7 %

I

Mean maximum corneal swelling at up to 240 min

6.5 %

II

Mean maximum corneal opacity

2.00

III

Mean fluorescein retention

3.00

IV

Other Observations

Test item was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Overall ICE Class

1xII 1xIII 1xIV

Interpretation of results:
other: no prediction can be made
Conclusions:
Based on this in vitro eye irritation in the isolated chicken eyes test with Ethoxylated phenol phosphate, the test item is not classified as a severe irritant and not classified as non-irritant.
Executive summary:

The eye irritancy potential of Ethoxylated phenol phosphate (100% purity) was investigated in the Isolated Chicken Eye Test (OECD 438) in vitro. The chicken eyes were treated for 10 seconds with the test substance. Afterwards, the eyes were rinsed with physiological saline. Irritation scores were taken at 0, 30, 75, 120, 180 and 240 minutes after rinsing. Based on the results obtained, the test item is not classified as a severe irritant and not classified as non-irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The potential of Ethoxylated Phenol Phosphate (100% purity) to induce skin irritation (OECD 431, 439) and eye irritation (OECD 437, 438) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered non-irritant to the skin. To assess the eye irritant potential of the target substance, the test item was tested in the Bovine Corneal Opacity & Permeability (BCOP) assay. The BCOP allows classification as “not classified” and “Eye Dam 1, H318” based on the In Vitro Irritancy Score (IVIS). Results which are in between both categories are identified as “No prediction can be made”. Based on the results from the BCOP test, no prediction can be made regarding the classification of the target substance. Subsequently, the In Vitro Eye Irritation Test in Isolated Chicken Eyes was conducted in accordance to OECD 438. This test can identify chemicals not requiring classification for eye irritation, or for serious eye damage (Eye Dam. 1, H318). Based on the results from this test, no prediction can be made regarding the classification of the target substance and the test item is not classified as a severe irritant and not classified as non-irritant. Therefore, the test item was considered not to be an ocular corrosive or severe irritant.

By assessing the results from both in vitro eye irritation tests in a weight-of-evidence approach, it can be concluded that classification as Eye Irrit 2, H319 is warranted as it was shown that the substance cannot be considered as non-irritant, but also not as inducing severe eye damage (Eye Dam 1, H318).