Reaction mass of dipotassium hydrogen phosphite and potassium dihydrogen phosphite

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

from September 18th to September 18th, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well described GLP study performed on the KH2PO3 monoconstituent substance. This form exists at lower pH (< 5.5) with a higher potential for irritation property

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD series on testing and assessment n. 113- Supplement to test Guidelines n. 437 and 438

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Potassium Hydrogen Phosphonate KH2PO3

IUPAC Name:

Potassium Hydrogen Phosphonate KH2PO3

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Molecular formula: KH2PO3
- Substance type: inorganic
- Physical state: white hygroscopic powder
- Storage condition of test material: ambient condition
Before the main assay, a solubility test was carried out resuspending the test item at 20% w/v (200 mg/mL) using physiological saline. Dissolution in the vehicle was immediate.
In the main assay the test item was dissolved in the same vehicle at the same concentration.

Test animals / tissue source

Species:

other: in vitro; bovine cornea

Details on test animals or tissues and environmental conditions:

Eyes were supplied by a butcher service.
- Source: Butcher Service s.r.l- Mattatoio n. 2067M, Viterbo, IT
- Age of animals: 6-12 months;
- Killing time: from 10:45 to 12:30 in the morning;
- Transport condition: at c.a 4°C in eye transport solution;

Test system

Amount / concentration applied:

MEDIA AND REAGENTS
Eyes transport solution: hanks balanced salt solution (HBSS);
Complete EMEM (with and without phenol red);
Sodium fluorescein solution

Duration of treatment / exposure:

Opacity determination: exposition of the corneas in horizontal position for 4 hours ± 5 minutes, incubated in a liquid bath at 32 ± 1 °C.
No post-exposure period.
Permeability: the corneas were horizontally incubated for approximately 90 minutes in a liquid batch at 32 ± 1 °C.

Observation period (in vivo):

Visual observation of corneas through chamber glasses and opacity determination.
Permeability determination after incubation

Details on study design:

PREPARATION OF CORNEAS
1) Eyes were examined for the presence of any defects (opacity, scratches or pitting of the corneal surface, vascularisation or pigmentation);
2) Cornea excision: each cornea with 2-3 mm of surrounding sclera was dissected from the eye using a scalpel, scissors and forceps and placed into a Petri dish containing HBSS (Hanks balanced salt solution);
3) Mounting of the cornea in the chamber with the endothelial surface placed in contact with the O-ring of the posterior part of the chamber in the correct position; the chamber was then filled with complete EMEM without phenol red maintained at 32 ± 1°C (posterior part of the chamber first to maintain convexity).
4) Equilibration: the corneas in their holders were incubated in a liquid bath at 32 ± 1°C at least for 1 hour to permit metabolic stabilisation.
At the end of the pre-dose incubation period, the two chambers were drained (anterior first) and re-filled with complete EMEM without phenol red maintained at approximately 32°C (posterior first).
5) Selection: basal opacity of corneas was determined by means of a calibrated meter, measuring difference in light transmission between the corneas placed into the beam paths versus air (opacitometer).
Cornea s with a basal value >= 7 arbitrary units were excluded from testing.
6) Determination of the mean opacity of the cornea;

METHOD
Test item: open chamber for a viscous substance;
Positive and negative controls: closed chamber method for free-flowing solutions.
The medium was completely removed from the anterior chamber. Corneas were treated with 0.75 mL of treatment volumes. After that, in the chambers the treatment was removed and corneas were exposed in horizontal position for 4h hours ± 5 minutes, incubated in a liquid bath at 32 ± 1°C. After a first washing with complete EMEM with phenol red a final wash with pre-warmed complete EMEM without phenol red was carried out. Finally, the anterior chamber was re-filled with pre-warmed complete EMEM without phenol red. Corneas were maintained in horizontal position, for further 2 hours ± 10 minutes, incubated in a liquid bath at 32 ± 1°C.

OBSERVATIONS
Visual observation through chamber glasses and pertinent observation recorded (e.g. tissue peeling/exfoliation, residual test substance, non-uniform opacity pattern etc.);

Opacity determination of all corneas: basal opacity of corneas was determined by means of a calibrated meter, measuring difference in light transmission between the corneas placed into the beam paths versus air (Opacitometer);

Permeability by treatment with fluorescein: 1 mL aliquot of 5 mg/mL sodium fluorescein solution in DPBS. The corneas were horizontally incubated for approximately 90 minutes in a liquid batch at 32 ± 1 °C. The quality ccontrols of the fluorescein solutions were assayed.

Permeability determination: measurement of the optical density of each sample using a spectrophotometer set at 490 nm.

Tissue retention.
At the end of the procedure, corneas were retained and fixed in 10% neutral buffered formalin.

Results and discussion

In vivo

Results

Irritation parameter:

other: in vitro irritancy score (IVIS)

Basis:

other: mean IVIS = mean opacity score + (15 x mean permeability OD 490 score)

Time point:

other: IVIS test item: 1.1; IVIS positive control: 89.5

Irritant / corrosive response data:

PREPARATION OF THE CORNEAS
A total of 14 corneas were processed (out of 16 delivered) for a final selection of the 9 required corneas according to basal opacity.
Eight corneas were actually assigned to treatment since the number of corneas was sufficient to cover all treatments scheduled in the experiment.

OBSERVATIONS
Negative control samples: no corrosion as the mean opacity values were homogeneous and in line with the expected values for this kind of control.
The positive control induced opacity of the whole cornea surface with a mean increase of the opacity value equal to 181.0. Severe opacity was noted in the two replicates at the macroscopic examination performed at the end of the 4-hour incubation period.
The test item increased very slightly the corneal opacity, being the calculated mean value 1.0. At the macroscopic observation the three corneas did not show any abnormality.On the other hand, cornea permeability was essentially unaffected when compared to that of negative control. The calculated mean permeability OD490 value was 0.0049.
The corneal permeability was also increased. The calculated mean permeability OD490 value was 2.8002.

Other effects:

Classification of the test item was performed according to the in vitro irritancy score:
> 55 corrosive or severely irritant;
In addition, the test item was evaluated also for the eye irritancy potential according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438.
0-3 Non eye irritation
3.1 - 25 Mild eye irritant
25.1 - 55 Moderate eye irritant

Any other information on results incl. tables

Table. 1

Treatment Code	Pre-test cornea number	Basal opacity	Mean group basal opacity
N1	13	0	0.3
N2	14	1
N3	3	0
P1	2	4	2.0
P2	1	0
A1	4	1	0.3
A2	5	0
A3	6	0
		Mean: 0.64 °
° the mean refers to the total number of selected pre-test corneas
^ showing a small lesion on the surface

TABLE 2 - Observation
Treatment	OPACITY								MACROSCOPIC OBSERVATIONS of the CORNEA	PERMEABILITY
			Basal Opacity Value	Assay Opacity Value	Value corrected (basal value)		Value Corrected (negative control)				OD490	Dilution factor	Value Corrected (dilution factor)	Value Corrected (negative control)
N1			0	3	3		N/A		No macroscopic changes		0,037	1	0,037	N/A
N2			1	-1	0	*	N/A		No macroscopic changes		0,014	1	0,014	N/A
N3			0	-1	0	*	N/A		No macroscopic changes		0,031	1	0,031	N/A
	Mean				1					Mean			0,0225
	SD				1,7					SD			0,012
	CV %				170					CV %			53,33
P1			4	185	181		180		Strongly opaque		1,85	1	1,85	1,828
P2			0	183	183		182		Strongly opaque		0,761	5	3,805	3,783
	Mean						181			Mean				2,805
	SD						1,4			SD				1,3824
	CV %						0,8			CV %				49,28
C1			1	1	0		0	*	No macroscopic changes		0,008	1	0,008*	0
C2			0	4	4		3		No macroscopic changes		0,042	1	0,042	0,02
C3			0	1	1		0		No macroscopic changes		0,014	1	0,014*	0
	Mean						1			Mean				0,0065
	SD						1,7			SD				0,0113
	CV %						170			CV %				173,85
* : Negative values assumed to be 0 for calculation purpose
N1-3: Negative control		P1-2: Positive control			C1-3: Test item

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria of the OECD Guideline 437: not corrosive/sever irritant to the eye. Criteria OECD supplement to Test Guidelines 437 and 438: no irritant effect to the eye Criteria used for interpretation of results: EU

Conclusions:

Very slight alterations of cornea opacity but not of permeability were recorded after treatment with the test item. According to the OECD Guideline no. 437, the test item is not to be classified as corrosive or severely irritant to the eye.
Moreover, according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438, there is no indication of an irritant effect to the eye
Very slight alteration of cornea opacity but not of permeability were recorded after treatment with the test item.. The IVIS score for the test item was 1.1.
According with OECD Guideline no. 437 the test item is not to be classified as corrosive or severe irritant to the eye. Moveover, according with OECD Supplement to Test Guidelines nos. 437 and 438 there is no indication of an irritant effect to the eye.

Executive summary:

The potential of the test item, Potassium hydrogen phosphonate, formula KH2PO3, to cause corrosion/severe irritation by using the Bovine Corneal Opacity and Permeability (BCOP) assay, was examined in agreement with OECD Guideline no. 437 (adopted on) and OECD Supplement to Test Guidelines nos. 437 and 438.

The test item was tested dissolved/suspended at 20% (w/v) in physiological saline (being a solid non surfactant) on the epithelial surface of three idoneous bovine corneas, for an exposure period of 4 hours.Positive and negative controls [at 20% (w/v) Imidazole solution in physiological saline and physiological saline alone, respectively] were concurrently tested in similar condition.

The mean opacity detected with an opacitometer at the end of the test item exposure period was 1.0, slightly higher than the negative control. After the determination of opacity, the epithelial surface was treated with a 0.5% solution of sodium fluorescein in DPBS for 90 minutes, to investigate alteration in cornea permeability.

Mean OD₄₉₀value of the corneas treated with the test item was essentially unaffected by treatment, as well as the negative control.

Negative and positive controls gave the expected results. 

The results obtained indicate that the test item induces slight changes in cornea opacity but not in corneal permeability under the reported experimental conditions. The calculated in vitro irritancy score (IVIS) for the test item is 1.1.

According to the OECD Guideline no. 437, the test item is not to be classified as corrosive or severely irritant to the eye. Moreover, according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438, there is no indication of an irritant effect to the eye.