Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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
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)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimethyl phosphate
EC Number:
208-144-8
EC Name:
Trimethyl phosphate
Cas Number:
512-56-1
Molecular formula:
C3H9O4P
IUPAC Name:
trimethyl phosphate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No. of test material: Sigma Aldrich, Saint Louis, USA; MKBX2207V
- Expiration date of the batch: 26/06/2022
- Purity test date: 97 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: yes

Test animals / tissue source

Species:
chicken

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 μL of test item was applied from micropipette onto the centre of the cornea, taking care not to damage or touch the cornea with the application equipment.
- Concentration: 100%

VEHICLE
- Amount(s) applied: 30 µl
- Concentration (if solution): positive control acetic acid 10 % (v/v) solution, negative control: isotonic saline [NaCl (9 g/L saline)]
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Number of animals or in vitro replicates:
3 test item replicates
3 positive control replicates
1 negative control
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Eyes Selection
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 fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with 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 eyeball 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.

Eyes Examination and Acclimatization 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), again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied 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 saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate numbers of eyes were selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic 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 using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm. Eyes 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 was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
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. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Changes in thickness were not observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NUMBER OF REPLICATES 3

NEGATIVE CONTROL USED - one replikate
NaCl (9 g/L saline)
Name: Sodium chloride
Supplier: lach:ner
Batch No.: PP/2015/05309
Expiry date: 11 December 2017
Storage condition: Room temperature
Diluted with ultra-pure water (prepared by Direct Q5 water purification system) in Toxi-Coop ZRT.

POSITIVE CONTROL USED - 3 replicates
Acetic acid 10% (v/v) solution
Name: Acetic acid (>=99.8%)
Supplier: SIGMA-ALDRICH
Batch No.: SZBE3160V
Retest date: 25 April 2018
Storage condition: Room temperature
The acetic acid (>=99.8%) was diluted with NaCl (9 g/L saline) to a final concentration of 10% (v/v). The formulation was prepared and used on the day of treatment.

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, one out of three test item treated eyes was held in horizontal position and 30 μL of test item was applied from micropipette onto the centre of the cornea, taking care not to damage or touch the cornea with the application equipment. This procedure was repeated with the remaining two test item treated eyes.
The three positive control eyes were treated with acetic acid 10 % (v/v) solution and one negative control eye was treated with isotonic saline [NaCl (9 g/L saline)] according the above procedure.

OBSERVATION PERIOD
The control and test eyes were evaluated at pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ±5 minutes were considered acceptable.
The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Scores were taken at any given time point.
- Damage to epithelium based on fluorescein retention:Scores were taken at any given time point
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: For the calculation of Maximum Swelling, small negative numbers for swelling (0 to -5%) following application are counted as zero. Large negative numbers (> -12 %) 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.

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score: 0, 0.5, 1, 2, 3, 4
- Mean fluorescein retention score at 30 minutes post-treatment: 0, 0.5, 1, 2, 3

DECISION CRITERIA: The conclusion on eye irritancy was based on the OECD guideline on quantitative assessments

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Remarks:
up to 75 min
Run / experiment:
mean
Value:
3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Remarks:
up to 240 min
Run / experiment:
mean
Value:
4
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
2.2
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
mean
Value:
1
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Positive and negative controls showed the expected results. The experiment was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
In this ICET, Trimethyl phosphate – 97% did not cause ocular corrosion or severe irritation in the enucleated chicken eyes.
In this in vitro eye irritation study, using the Isolated Chicken Eye model with Trimethyl phosphate – 97%, ocular corrosion and severe irritation potential was not observed. The overall ICE score was 1xI, 1xII, 1xIII.
According to the guideline OECD 438, Trimethyl phosphate overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.