Magnesium hexafluorosilicate

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

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium hexafluorosilicate hexahydrate

Cas Number:

18972-56-0

Molecular formula:

MgSiF6·6H2O

IUPAC Name:

Magnesium hexafluorosilicate hexahydrate

Test material form:

other: crystalline powder

Test animals / tissue source

Species:

other: bovine

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: bovine
- Eye transport solution: Hanks balanced salt solution (HBSS) Modified [Gibco Cat. no. 14025 (batch no. 1150029)] supplemented with Penicillin sulphate (Sigma batch no. BCBD3925V) and Streptomycin sulphate (Sigma batch no. 079K14102V) both at the final concentration of 100 IU/mL.
Complete EMEM* (also named: Minimum Essential Medium Eagle's (MEM) Modified or MEM or Eagle’s MEM.):
- without phenol red: EMEM* Gibco (Invitrogen) Cat. no. 51200 (batch no. 1150177) supplemented with 1% (v/v) Foetal Bovine Serum (FBS - ATCC - batch no. 30001205)
- with phenol red: EMEM* Gibco (Invitrogen) Cat. no. 21090 (batch no. 1098654) supplemented with 1% (v/v) Foetal Bovine Serum (FBS - ATCC - batch no. 30001205)
These media were prewarmed in a water bath at 32°C during the experimental procedure.
- Sodium fluorescein solution : Sigma Cat. no. F-6377 (batch no. 079K0141). This was formulated to give 5 mg/mL solutions in DPBS [Gibco Cat. No. 14190 (batch no. 1098734)].
- Transport condition: Maintained in eyes transport solution at approximately 4ºC

COLLECTION OF EYES
Slaughter house: Butcher Service s.r.l.- Mattatoio no. 2067 M St. Teverina Km. 7800 - 01100 Viterbo
Age of animals : 6-12 months
Killing time: From 9:00 to 11:30 in the morning.
Transport condition: Maintained in eyes transport solution at approximately 4°C.
Approximate test starting time: 13:50

PREPARATION AND SELECTION OF CORNEAS
Examination: Eyes were examined for the presence of any defects which could render the eye unsuitable for use (opacity, scratches or pitting of the corneal surface, vascularisation or pigmentation).
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.
Mounting in the chamber: Each cornea was mounted into a prewarmed testing chamber (see Figure 1) with the endothelial surface of the cornea placed in contact with the O-ring of the posterior part of the chamber. Care was taken to ensure the correct position of the cornea minimizing the presence of pigmented area inside the O-ring and avoiding movements which would damage the cornea. 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).
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).
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). During measurement, attention was paid to keep corneas away from the bath for the minimal time needed.
Corneas with a basal value ≥ 7 arbitrary units are excluded from testing.
The mean opacity of the remaining corneas was determined. Corneas were distributed in treatment groups starting from those nearest to the mean value for the negative control group and trying to approximately maintain opacity values similar to the mean of the negative control group in the treatment and positive control groups.

Test system

Vehicle:

physiological saline

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 20 % (w/v) being the test item a solid non surfactant

VEHICLE
- Concentration (if solution): 0.9%% NaCl (Physiological saline)
- batch no. (if required): 10I0208

Duration of treatment / exposure:

4h +/- 10 min

Observation period (in vivo):

There was no post-exposure period.

Number of animals or in vitro replicates:

9 corneas

Details on study design:

CONTROL ITEMS
Positive control item: 20% (w/v) Imidazole (Sigma Cat. no.: 56749; CAS: 288-32-4, batch no.: 0001422794) solution in physiological saline (Bieffe Medital, batch no. 10I0208), being the test item a solid. A certificate of analysis is presented in Addendum II.
Negative control item: Physiological saline (0.9% NaCl; BieffeMedital batch no. 10I0208) as a control of the test system.

Positive and negative control items were obtained commercially and characterised by their labelling. Determination of the stability and concentration of solutions of the positive control was not undertaken since it is sufficient to provide evidence for the correct expected response of the test system to it.

ANALYSIS OF DATA

For each test point, the mean value of opacity obtained after exposure was calculated. As appropriate, negative control was subtracted from the opacity of the test item or positive control.
The mean value of permeability, expressed as optical density units, was also calculated and corrected against the mean control value.
The in vitro irritancy score (IVIS) of the test item was calculated from these values in the following manner:
IVIS = mean opacity score + (15 x mean Permeability OD490 score)
Classification of the test item was performed according to the in vitro irritancy score:
Value Classification
> 55.1 Corrosive or severe 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:

Value Classification
0-3 Non eye irritant
3.1-25 Mild eye irritant
25.1- 55 Moderate eye irritant

TOOL USED TO ASSESS SCORE: fluorescein

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: In vitro irritancy score (IVIS)

Basis:

mean

Score:

ca. 109.3

Results 2

Irritation parameter:

other: OD490 (corneal permeability)

Basis:

mean

Score:

ca. 1.636

Irritant / corrosive response data:

According to OECD Guideline no. 437, the test item should be classified as corrosive or serverely irritant to eye.

Any other information on results incl. tables

PREPARATION OF CORNEAS

Once underwent the first examination, a total of 9 corneas were processed (out of 10 delivered) for a final selection of the 9 required corneas according to basal opacity.

The measured basal opacity values and assignment to treatment groups of the processed corneas are presented in Table 1.

Treatment Code	Pre-test cornea number	Basal opacity	Mean group basal opacity
N1	1	2	1.0
N2	2	1
N3	5	0
P1	3	1	1.0
P2	7	0
P3	10	2
C1	4	1	1.0
C2	6	1
C3	8	1
		Mean: 1.0 °

OBSERVATIONS

Details of measurements carried out at the observations are reported in Table 2.

STUDY NO.: 93360

 

Treatment code		OPACITY						MACROSCOPIC OBSERVATIONS			PERMEABILITY
		Basal Opacity Value	Assay Opacity Value	Value corrected (basal value)		Value Corrected (negative control)					OD490	Dilution factor	Value Corrected (diluition factor)	Value Corrected (negative control)
N1		2	2	0		N/A		No macroscopic changes			0.013	1	0.013	N/A
N2		1	2	1		N/A		No macroscopic changes			0.013	1	0.013	N/A
N3		0	2	2		N/A		No macroscopic changes			0.016	1	0.016	N/A
	Mean			1.0						Mean			0.0140
	SD			1.0						SD			N/A
	CV %			100.0						CV %			N/A
IVIS:	1.2
P1		1	47	46		45		Opacity			1.148	1	1.148	1.134
P2		0	45	45		44		Opacity			1.208	2	2.416	2.402
P3		2	35	33		32		Opacity			1.387	1	1.387	1.373
	Mean					40.3				Mean				1.636
	SD					7.2				SD				0.674
	CV %					17.9				CV %				41.18
IVIS:	64.8
C1		1	116	115		114		Opacity			0.003	1	0.000	-0.014	*
C2		1	93	92		91		Opacity			0.008	1	0.000	-0.014	*
C3		1	125	124		123		Opacity			0.010	1	0.000	-0.014	*
	Mean					109.3				Mean				0.000
	SD					16.5				SD				N/A
	CV %					15.1				CV %				N/A
IVIS:	109.3
N/A:not applicable			*assumed to be 0 for calculation

The negative control treatments did not induce corrosion as the mean opacity and permeability values were homogeneous and in line with the expected values for this kind of control.

The test item induced opacity of the whole cornea surface, with a mean increase of the opacity value equal to 109.3. This was confirmed at the macroscopic observation in which the three corneas showed visible opacity.

With reference to the cornea permeability, this was not affected when compared to that of negative control.

The positive control induced opacity of the whole cornea surface with a mean increase of the opacity value equal to 40.3. Opacity was noted in the three replicates at the end of the 4-hour post-incubation period.

The corneal permeability was also increased. The calculated mean permeability OD490 value was 1.636.

IVIS CALCULATION

The IVIS was calculated as follows:

IVIStest item = 109.3

IVISpositive control = 64.8

The test is therefore considered as valid.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The potential of the test item, Magnesium Hexafluorosilicate hexahydrated (MgSiF6), to be severely irritant or corrosive to the eye, was measured by its ability to induce opacity and increase permeability in isolated bovine corneas.

Alterations of cornea opacity were recorded during the study when compared to those of negative control. No significant alterations of cornea permeability were noted. The negative and positive controls gave the expected results. The test is therefore considered as valid.

According to the OECD Guideline no. 437, the test item should be classified as corrosive or severely irritant to the eye.

Executive summary:

The potential of the test item, Magnesium Hexafluorosilicate Hexahydrated (MgSiF6·6H2O), 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 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 [a 20% (w/v) Imidazole solution in physiological saline and physiological saline alone, respectively] were concurrently tested in similar conditions.               

The mean opacity detected with an opacitometer at the end of the test item exposure period was 109.3.  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 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 induced changes in cornea opacity, but not in permeability under the reported experimental conditions. The calculated in vitro irritancy score (IVIS) for the test item is 109.3.  

According to the OECD Guideline no. 437, the test item should be classified as corrosive or severely irritant to the eye.