Diallyl hexahydrophthalate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 December 2016 - 16 August 2017

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:

2017

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

Remarks:

The draft report was issued later than stated in the Study Plan due to organisational changes (14 Feb 2017) where S. Gáty became responsible for QA (15 Feb 2017). These deviations had no adverse affect on the integrity or validity of the study.

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

Remarks:

The draft report was issued later than stated in the Study Plan due to organisational changes (14 Feb 2017) where S. Gáty became responsible for QA (15 Feb 2017). These deviations had no adverse affect on the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Diallyl hexahydrophthalate

EC Number:

237-580-1

EC Name:

Diallyl hexahydrophthalate

Cas Number:

13846-31-6

Molecular formula:

C14H20O4

IUPAC Name:

1,2-bis(prop-2-en-1-yl) cyclohexane-1,2-dicarboxylate

Test material form:

liquid

Remarks:

Colourless

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test material: MDAC
- Appearence: Colourless liquid
- Source: Osaka Soda Co., Ltd
- Batch/lot No: 40201
- Expiration date of the lot/batch: 26 January 2019
- Purity test date: 99.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70% Relative Humidity (RH)), protected from light
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: MDAC was soluble in physiological saline (30 μL test item in 1 mL saline). Assumed stable for the duration of the study
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út 129., Hungary). Chicken heads were obtained from a veterinary-inspected, commercial slaughter-house, processing chickens for human consumption.
- Number of animal heads: 4/5 chicken heads per box. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box and closed.
- Characteristics of donor animals (e.g. age, sex, weight): Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were collected by a slaughter house technician and heads transported at ambient temperature to CiToxLAB Hungary Ltd. at the earliest convenience (i.e. within 2-hours)
- Time interval prior to initiating testing: The heads were received at CiToxLAB and processed within approximately 2 hours of collection. For selected eyes deemed appropriate for testing, acclimatization started and was conducted for approximately 45 to 60 minutes.
- indication of any existing defects or lesions in ocular tissue samples: No, the appropriate number of eyes were examined (with a slit lamp microscope) to ensure that all were in good condition and suitable for study use.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of test item was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with test substance. The test substance was applied as supplied, no formulation was required.

VEHICLE
- Amount(s) applied (volume or weight with unit): 30 μL of physiological saline was applied to the negative control eye.
- Concentration (if solution): Physiological saline (0.9% (w/v) NaCl)
- Lot number: 62351Y05-2
- Manufacturer: B. Braun Pharmaceuticals SA
- Expiry Date: 31 May 2019
- Storage condition: Room temperature

Duration of treatment / exposure:

The time of application was noted and after an exposure period of 10 seconds, 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 test item.

Duration of post- treatment incubation (in vitro):

240 minutes. The control eyes and test eyes were evaluated pre-treatment and at ca. 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

Details on study design:

SELECTION OF ISOLATED EYES
After removing the head from the plastic box, it was placed on soft paper. The eyelids were carefully cut away using scissors avoiding any damage to 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. The fluorescein treated cornea was then 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 acceptable for study use, the eyeball was carefully removed from the orbit.

PREPARATION OF ISOLATED 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 the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent corneal distortion and subsequent corneal opacity. Once removed, the eye was placed onto damp paper and the nictitating membrane cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box to maintain appropriate humidity.

EQUILIBRATION (Acclimitisation) AND BASELINE RECORDINGS
Prepared eyes were placed in thier respective steel clamp with the cornea positioned vertically with the eye in (as per placement within the chicken head). Too much pressure on eyes was avoided when clamping. 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 each eye was positioned such 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 any experimental adjustiments or examinations.

The appropriate number of eyes were selected and after being placed in the superfusion apparatus. They were then examined again using a slit lamp microscope to ensure they were acceptable for use. The focus was adjusted to clearly see the physiological saline 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 appropriate for testing, acclimatization started, this was for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were controlled at a temperature (32±1.5°C) during the acclimatisation and treatment periods.

At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. Cornea thickness 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 of this study. 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 suitable for study use. Fluorescein 10% (lot No. 250817F, Manufacturer. Alcon, Expiry date. 31 May 2017) was diluted with physiological saline to a final concentration of 2% (w/v). The formulation was allocated an expiry date of 2 Feb 2017.

QUALITY CHECK OF THE ISOLATED CORNEAS:
The fluorescein treated cornea was examined using a hand-held slit lamp or slit lamp microscope, with the eye still within the chicken head, this ensured the cornea was not damaged. If the cornea was judged adequate for study use the eyeball was carefully removed from the orbit. The appropriate number of eyes was thereafter selected and placed in the superfusion apparatus. They were then examined again to ensure studt acceptability. The focus was adjusted to see clearly the physiological saline 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.

NUMBER OF REPLICATES:
Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

NEGATIVE CONTROL USED:
Physiological saline solution (0.9% (w/v) NaCl). Expiry date 31 May 2019.

POSITIVE CONTROL USED:
Benzalkonium chloride solution, 50% in water diluted to acheive a final concentration of 5% (w/v). Allocated expiry date for the positve control formulation was Sep 2017.

APPLICATION DOSE AND EXPOSURE TIME:
30 μL of test item was applied to the entire surface of the cornea and exposed for a period of 10 seconds from the end of applicationn to the cornea surface

TREATMENT METHOD: Closed clamping chamber

POST-INCUBATION PERIOD: yes. 240 minutes. The control eyes and test eyes were evaluated pre-treatment and at Ca.30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The test item was rinsed thoroughly with 20 mL physiological saline at ambient temperature. Care was taken to not damage the cornea but attempting to remove all residual test item.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal thickness and opacity were measured and scored at all time points (30, 75, 120, 180 and 240 minutes after exposure) Fluorescein
retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit BP 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.

SCORING SYSTEM:
The mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change are given below. The conclusion on eye irritancy was based on the OECD guideline quantitative assessments, as detailed below:

Mean corneal swelling (%). No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes.

Corneal Swelling (%) ICE Class
0 to 5 I
>5 to 12 II
>12 to 18 (>75 min after treatment) II
>12 to 18 (≤75 min after treatment) III
>18 to 26 III
>26 to 32 ( 75 min after treatment) III
>26 to 32 (≤75 min after treatment) IV
>32 IV

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).

- Mean maximum opacity score. No significant corneal opacity change (severity 0.5) was noted on two eyes.

Mean Maximum Opacity Score ICE Class
0.0 – 0.5 I
0.6 – 1.5 II
1.6 – 2.5 III
2.6 – 4.0 IV

- Mean fluorescein retention score at 30 minutes post-treatment. No significant fluorescein retention change (severity 0.5) was observed on one eye. No other corneal effect was observed.

Mean Fluorescein Retention Score at 30 minutes post-treatment ICE Class
0.0 – 0.5 I
0.6 – 1.5 II
1.6 – 2.5 III
2.6 – 3.0 IV

DECISION CRITERIA: In Vitro Irritancy Score (IVIS): The irritation effects of the test item (MDAC) were evaluated according to the OECD No. 438 (26 July 2013). The test as described in OECD 438 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).

DECISION CRITERIA: The conclusion on eye irritancy was based on the OECD guideline quantitative assessments. The mean values of treated eyes for maximum corneal thickness, corneal opacity and fluorescein retention are documented in line OECD 438 classification requirements.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum swelling up to 240 minutes

Value:

ca. 1.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Mean maximum corneal opacity upto 240 minutes

Value:

ca. 0.33

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean fluorescein retention upto 30 minutes

Value:

ca. 0.17

Vehicle controls validity:

valid

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

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The results from all eyes used in the study met the quality control standards. Thenegative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control results were in line with historical data.
- Acceptance criteria met for positive control: The positive control results were in line with historical data.

The results from all eyes used met the quality control standards. The negative control and positive control results were in line with historical data. This experiment was
considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant corneal opacity change (severity 0.5) was noted on two eyes. No significant fluorescein retention change (severity 0.5) was observed on one eye. No other corneal effects were observed.

Based on this in vitro eye irritation in the isolated chicken eyes test with MDAC, the test item is non-irritant. UN GHS Classification is No Category.
.