Phosphoric acid, C8-10-alkyl esters

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 May 2016 to 22 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Confidential

IUPAC Name:

Confidential

Test material form:

liquid

Details on test material:

- Appearance/physical state: Amber liquid
- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%)

Test animals / tissue source

Species:

chicken

Strain:

other: COBB 500 in Experiment I and ROSS 308 in Experiment II

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Approximately 30 μL of test item

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

Three each in experiments I and II

Details on study design:

INTRODUCTION
- The Enucleated Eye Test with isolated eyes of chickens is a well validated and accepted in vitro test system. It has been recognised as a valuable alternative to the Draize eye irritation test, because it represents a test system nearest to the in vivo test, without the need to use live animals. It can also be used as a screening tool for corrosivity/severe irritancy to avoid unacceptable effects in vivo. In the Isolated Chicken Eye Test (ICET) the test compound is applied in one single dose onto the cornea of isolated eyes. Chicken heads are obtained from a veterinary-inspected, commercial slaughter-house, processing chickens for human consumption.
- This method can provide detailed information about the effects of test items on the cornea, and can be used to identify chemicals not requiring classification for eye irritation, or for serious eye damage, as defined by the UN GHS (UN GHS nonclassified or UN GHS Category 1). The test is described in OECD No. 438 and is approved by international regulatory agencies as a replacement for the identification of non-irritant, corrosives/severe irritants in the in vivo Rabbit Eye Assay (OECD No. 405).
- The procedure was first performed on 3 test item treated eyes, 3 positive control eyes and 1 negative control eye (Experiment I.) then on a further 3 test item treated eyes, 3 positive control eyes, 1 negative control eye and 1 negative control eye with more excessive rinsing and extension observation time (Experiment II.) due to uncertainty for part of the results in the first run.

TEST ITEM SOLUBILITY AND FORMULATION
- Solubility of the test item in physiological saline was tested prior to the Experiment I (30 μL test item in 1 mL saline). The test item did not dissolve in physiological saline.
- The test item was applied as supplied, no formulation was required.

POSITIVE CONTROL
- The material was diluted with distilled water (Supplier: Teva Co., Lot number: 6820914, Expiry date: September 2017 in Experiment I and Lot number: 7170914, Expiry date: September 2017 in Experiment II) to achieve a final concentration of 5% (w/v).

SUBSIDIARY MATERIAL
- Experiment I: Fluorescein sodium (Merck; lot number K46664387; expiry date 31 May 2020). This material was mixed with physiological saline (Manufacturer: B. Braun Melsungen AG, Lot number: 52532Y05-2, Expiry date: 31 May 2018) to achieve a final concentration of 2% (w/v). The resulted solution was stored at room temperature (Dispensary code: S43100, Expiry date: 02 June 2016.).
- Experiment II: Fluorescein sodium 10 % w/v (ALCON; lot number 250817F; expiry date 31 May 2017). This fluorescein solution was mixed with physiological saline (Manufacturer: B. Braun Pharmaceuticals SA, Batch number: 52532Y05-2; Expiry date: 31 May 2018) to achieve a final concentration of 2% (w/v). The resulted solution was stored at room temperature (Dispensary code: S43102, Expiry date 07 July 2016).

CHICKEN HEADS COLLECTION AND TRANSPORT
- Source: TARAVIS KFT, 9600 Sárvár, Rábasömjéni út.129., Hungary.
- 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 approximately 2 hours of collection.

SELECTION OF EYES
- After removing the head from the plastic box, it was put on soft paper.
- The eyelids were carefully cut away with scissors, avoiding damaging the cornea.
- One small drop of 2 % (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. - If the cornea was in good condition, the eyeball was carefully removed from the orbit.

PREPARATION OF EYES
- The eye ball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity.
- Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EYE EXAMINATION AND ACCLIMATISATION TIME
- 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) avoiding 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 ( > 0.5) or corneal opacity score (> 0.5) were rejected. The cornea thickness was measured and 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, the acclimatisation started and was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32 ± 1.5°C) during the acclimatisation and treatment periods.

IDENTIFICATION
- The eyes were identified by chamber number, marked on the door of the chamber.

BASELINE ASSESSMENTS
- At the end of the acclimatisation period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye.
- The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time.
- No changes in thickness (0.0%) were observed in the eyes.
- Following the equilibration period, the fluorescein retention was measured.
- Baseline values were required to evaluate any potential test item related effect after treatment.
- All eyes were considered to be suitable for the assay.

TREATMENT
- 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.
- Approximately 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 and negative control eye with more excessive rinsing were treated with 30 μL of physiological saline; positive control eyes were treated with 30 μL 5% (w/v) Benzalkonium chloride solution.

TEST ITEM REMOVAL
- The time of application was observed, 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 at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test item if possible.
- Additional gentle rinsing with 20 mL saline was performed at each time point when remaining test item or control material was observed on the cornea.
- Note: More excessive rinsing with 31.7 °C physiological saline was applied in Experiment II.

OBSERVATION AND ASSESSMENT OF CORNEAL EFFECTS
- 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, using a Haag-Streit BP 900 slit-lamp microscope.

EVALUATION
- Corneal swelling was calculated using the formulae CS at time t = (CT at time t – CT at t=0 / CT at t = ) x 100 and Mean CS at time t = (FECS at time t + SECS at time t + TECS at time t) / 3 where CS = cornea swelling; CT = cornea thickness; FECS at time t = first eye cornea swelling at a given time point; SECS at time t = second eye cornea swelling at a given time-point; TECS at time t = third eye cornea swelling at a given time-point.
- Small negative numbers for swelling (0 to -5%) following application are evaluated as class I. Large negative numbers (> 12% below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5% to -12% are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).
- Cornea opacity was calculated according to the formulae ACO at time t = CO at time t – CO at t=0 and Mean ΔCO max = (FECO max 30 min to 240 min + SECO max 30 min to 240 min + TECO max 30 min to 240 min) / 3 where CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the post treatment rinse; CO at t=0 = baseline cornea opacity; ΔCO at time t = difference between cornea opacity at t time and cornea opacity baseline; FECO = first eye cornea opacity; SECO = second eye cornea opacity; TECO= third eye cornea opacity; max 30min to 240min = maximum opacity of the individual eye at 30 to 240 minutes minus
baseline cornea opacity of the individual eye.
- Fluorescein retention was calculated according to the formulae ΔFR at time t = FR at time t – FR at t=0 and Mean ΔFR = (FEFR 30 min + SEFR 30 min + TEFR 30 min) / 3 where FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse; FR at t=0 = baseline fluorescein retention; ΔFR at time t = difference between fluorescein retention at t time and fluorescein retention baseline; FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention; SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention; TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention.

RETENTION OF CHICKEN EYES
- At the end of the procedure, the corneas from the eyes were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10% neutral buffered formalin, Manufacturer: Reanal, Batch number: KTM14051, Expiry date: October 2017 in Experiment I. and Batch number: KTM15011, Expiry date: February 2018 in Experiment II.) was used for potential histopathology and stored at room temperature.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: overall ICE class

Run / experiment:

Experiment I

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 1 x III, 1 x IV

Remarks:

test item

Results 2

Irritation parameter:

other: overall ICE class

Run / experiment:

Experiment II

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 2 x III, 1 x IV

Remarks:

test item

Other effects / acceptance of results:

TEST ITEM
- Individual data for Experiments I and II are shown in Tables 1.1 and 1.2 (see Appendix 1, attached).
- The mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change are attached for Experiments I and II. The conclusion on eye irritancy was based on the OECD guideline quantitative assessments, shown in Appendix 2 (attached).
- Details of data interpretation for Isolated Chicken Eye (ICE) Class are given in Appendix 2 and 3 (attached). The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity and the mean fluorescein retention ICE classes are used for EC and GHS classification.
- In Experiment I, test item was stuck on all cornea surfaces (confluent layer) during the full post treatment period and could not be rinsed off and all three cornea surfaces were not cleared at 240 minutes after the post-treatment rinse, and as a result, the corneal opacity, the corneal swelling and the fluorescein retention could not be properly evaluated.
- Due to the equivocal results, an additional experiment was performed for clarification as agreed by the sponsor. The experimental conditions were the same as in the first test, except for more excessive rinsing with physiological saline (31.7 °C) and an extension of the observation time. Moreover an additional control (standard controls and one negative control with similar excessive rinsing) demonstrated that the additional washing had no adverse effect.
- In Experiment II, all three cornea surfaces were not cleared fully at 300 minutes after the post-treatment rinse, however the corneal thickness, corneal opacity and fluorescein retention was assessed on the clear areas of eyes. The areas of test item stuck to the cornea could mask corneal effects underneath the residues and could also potentially result in mechanical damage in vivo. Based on this the scores situated the test item as less than, but close to, the cut-off for a severe irritant. The test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that an in vivo study is required for proper classification.
- A summary table showing UN GHS classification information relating to the test item is attached.

POSITIVE CONTROL
- Individual data for Experiments I and II are shown in Tables 1.3 and 1.4 (see Appendix 1, attached).
- The positive controls 5% (w/v) Benzalkonium chloride solutions were classified as severely irritating, UN GHS Classification: Category 1 (see results for Experiments I and II, attached).

NEGATIVE CONTROL
- Individual data for Experiments I and II are shown in Tables 1.5, 1.6 and 1.7 (see Appendix 1, attached).
- The negative controls Physiological saline solutions were classified as non-irritating, UN GHS Classification: Non-classified (see results for Experiments 1 and II, attached).

VALIDITY OF THE TEST
- Historical control data is attached.
- The results from all eyes used met the quality control standards. The negative control and positive control results were in line with historic data. This experiment was considered to be valid.
- For the classification, the guideline clearly says that alcohol containing test items may be over-predicted. A more reliable result may be obtained following histopathology evaluation of the corneas.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item was not classified as a severe irritant and not classified as non-irritant using the in vitro isolated chicken eye test method. It was therefore concluded that an in vivo study was required.

Executive summary:

GUIDELINE

An in vitro eye irritation study of the test item was performed in isolated chicken eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 (26 July 2013).

 

METHODS

After the zero reference measurements, the eye was held in horizontal position and approximately 30 μL of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 μL Benzalkonium chloride 5% solution. The negative control eye was treated with 30 μL of physiological saline (9% (w/v) NaCl solution). Examinations in Experiment I were three test item treated eyes, three positive control treated eyes and one negative control treated eye. In Experiment II, three test item treated eyes with more excessive rinsing, three positive control treated eyes, one negative control treated eye and one negative control treated eye with more excessive rinsing were examined. The observation time was also extended in Experiment II.

 

RESULTS

Results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

 

Test item was stuck on all cornea surfaces (confluent layer) during the full post treatment period and could not be rinsed off (all three cornea surfaces were not cleared at 240 minutes after the post-treatment rinse) and, as a result, the corneal opacity, the corneal swelling and the fluorescein retention could not be properly evaluated.

 

Due to the equivocal results, an additional experiment was performed for clarification as agreed by the sponsor.The experimental conditions were the same as in the first test, except for more excessive rinsing with physiological saline (31.7 °C) and an extension of the observation time. Moreover an additional control (standard controls and one negative control with similar excessive rinsing) demonstrated that the additional washing had no adverse effect.

 

Experiment I: Severe corneal swelling was observed during the four-hour observation period on test item treated eyes. Cornea opacity change could not be evaluated. Moderate fluorescein retention change (severity 2) was noted on three eyes. Test item was stuck on all cornea surfaces (confluent layer) after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

 

Experiment II: Moderate corneal swelling was observed during the five-hour observation period on test item treated eyes. Moderate cornea opacity change (severity 1 or 2) was observed on three eyes. Severe fluorescein retention change (severity 2 or 3) was noted on three eyes. These scores situate the test item as below, but close to, the cut-off for a severe irritant. Relatively large areas of the test item could be rinsed off, but small areas of the test item had material still stuck on all cornea surfaces even with after the post- excessive rinsing. All three cornea surfaces were not cleared fully at 300 minutes after the post-treatment rinse. The areas of test item stuck to the cornea could mask corneal effects underneath the particles and could also potentially result in mechanical damage in vivo.

 

CONCLUSION

The test item was not classified as a severe irritant and not classified as non-irritant using the in vitro isolated chicken eye test method. It was therefore concluded that an in vivo study was required.