Phosphonic acid, calcium salt (2:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July 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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phosphonic acid, calcium salt (2:1)

Cas Number:

13780-04-6

Molecular formula:

Ca·2H3O3P

IUPAC Name:

Phosphonic acid, calcium salt (2:1)

impurity 1

Reference substance name:

Phosphonic acid

EC Number:

237-066-7

EC Name:

Phosphonic acid

Cas Number:

13598-36-2

Molecular formula:

H3O3P

IUPAC Name:

Phosphorous acid

impurity 2

Reference substance name:

Minor impurities not relevant for classification

IUPAC Name:

Minor impurities not relevant for classification

additive 1

Reference substance name:

Silicon dioxide

EC Number:

231-545-4

EC Name:

Silicon dioxide

Cas Number:

7631-86-9

Molecular formula:

O2Si

IUPAC Name:

dioxosilane

Test material form:

solid: particulate/powder

Details on test material:

- Density: 2.13 g/cm3- Moisture content: 4.0% (w/w)

Test animals / tissue source

Species:

chicken

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg per eye

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYESThe eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay. The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 24 July 2017 at 8:25 am.Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.The eyes were enucleated at Phycher on 24 July 2017 at 10:15 am.The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.3°C and 32.9°C.EQUILIBRATION AND BASELINE RECORDINGSAfter being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.Once all eyes had been examined and approved, the eyes were incubated between 45 and 65 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.NUMBER OF REPLICATES: 3NEGATIVE CONTROL USEDphysiological saline – Dutscher Batch No. 3012575; expiry date of batch: December 2018SOLVENT CONTROL USEDnot applicablePOSITIVE CONTROL USED sodium hydroxide CAS No.:1310-73-2 – Sigma, Batch No. MKBP7805VAPPLICATION DOSE AND EXPOSURE TIMEImmediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, as supplied, to the cornea such that the entire surface of the cornea was evenly covered with the test item.Then the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.OBSERVATION PERIODTreated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.REMOVAL OF TEST SUBSTANCE- Volume and washing procedure after exposure period: the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature.- Indicate any deviation from test procedure in the Guideline: noneMETHODS FOR MEASURED ENDPOINTS: All observations of the cornea and measurement of corneal thickness were performed using a Haag- Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness, the slit-width was set at 91⁄2, equalling 0.095 mm. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which was determined only at pretreatment and 30 minutes after test item exposure) were determined at each of the above time points.SCORING SYSTEM:- Mean corneal swelling (%): Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope. It was expressed as a percentage and was calculated from corneal thickness measurements. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item.- Mean maximum opacity score: Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item.- Mean fluorescein retention score at 30 minutes post-treatment: The mean fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. Yes

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

maximal mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class I

Results 2

Irritation parameter:

fluorescein retention score

Remarks:

mean

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class IV

Results 3

Irritation parameter:

percent corneal swelling

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class I

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

inconclusive for classification

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item CALCIUM DIHYDROGENPHOSPHITE is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.