Ammonium magnesium orthophosphate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-01-28 - 2013-02-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The GLP study was conducted according to an internationally accepted guideline. All study parameters are given in detail.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Ocular Irritation Protocol: Neat Method (MTT ET-50) for use with EpiOcular TM Tis-sues, Rev: 17. Jan. 2012 by MatTek

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium magnesium orthophosphate

EC Number:

232-075-2

EC Name:

Ammonium magnesium orthophosphate

Cas Number:

7785-21-9

Molecular formula:

H3-N.H3-O4-P.Mg

IUPAC Name:

ammonium magnesium orthophosphate

Test material form:

solid: crystalline

Details on test material:

Name Magnesium Ammonium Phosphate (MAP, "Berliner Pflanze")
Batch no. 10.10.2012
Appearance solid (crystals)
Composition P 19.3 %; Mg 14.2 %; NH4 10.11 %; SiO2 7% (dry matter)
CAS No. 7785-21-9
EINECS-No. 232-075-2
Molecular formula NH4MgPO4
Molecular weight 137.3148 g/mol
Purity 81% TR

Test animals / tissue source

Species:

other: not applicable: in vitro method

Strain:

other: not applicable: in vitro method

Details on test animals or tissues and environmental conditions:

not applicable: in vitro method

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable: in vitro method

Amount / concentration applied:

3 minutes 30 minutes 60 minutes
102.0 mg 102.3 mg 99.8 mg
103.2 mg 104.0 mg 101.1 mg

Duration of treatment / exposure:

3, 30, 60 minutes

Number of animals or in vitro replicates:

not applicable: in vitro method

Details on study design:

On the day of the start of the experiment, the MTT concentrate was thawed. The concen-trate was diluted with the MTT solvent and the solution was stored at 4 °C in the dark.
The assay medium was warmed in the water bath to 37 °C.
The solid test item was ground.
Six sterile 6-well-plates were prepared with 0.9 mL assay medium in each well. The inserts containing the tissues were transferred to the wells using sterile forceps and the 6-well-plates were set into the incubator at 37 ± 1 °C, 5 ± 1 % CO2 for one hour pre-incubation.
Two 24-well-plates were prepared as holding plates. 12 wells of each plate were filled with 300 µL assay medium, the other 12 wells with 300 µL MTT medium. One additional plate was left empty. The plates were stored in the incubator.
After pre-incubation, the assay medium was replaced by fresh assay medium and the test is started. At the beginning of the experiment, a stop watch was started.
First the negative control (exposure time 60 min) was applied to two tissues, using 100 µL H2O demin. per tissue. Next, 100 mg of the solid test item were applied together with 30 µL H2O to two tissues (exposure time 60 min.). Then, two wells were used as positive control with 100 µL 0.3% Triton X 100 solution (exposure time 45 min.).After that 100 mg of the solid test item were applied together with 30 µL H2O to two tissues for 30 min exposure. Next, two tissues were used as positive control again with 100 µL 0.3 %Triton X 100 solution for 15 min. exposure. Last, 100 mg of the solid test item were applied together with 30 µL H2O in duplicate for a 3 min exposure time.
After the respective incubation times, the inserts were removed from the plates using ster-ile forceps. The inserts were thoroughly rinsed with DPBS and soaked for 10 minutes in 5 mL assay medium to remove any traces of the test item. They were finally blotted with sterile cellulose tissue for drying the inserts and set into the respective holding plate, using the wells containing assay medium. After transfer of all inserts, they were moved to the wells containing MTT solution, blotting the bottom with cellulose tissue again before setting the insert into the MTT well. The tissues were incubated with MTT medium for three hours. After this time, the MTT medium was aspirated and replaced by DPBS buffer. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into an empty 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then covered with Parafilm® and left to stand overnight at room temperature.
On the next day, the inserts in which formazan had been produced were pierced with an injection needle, taking care that all colour was extracted.
The inserts were then discarded and the content of the 24-well plate was thoroughly mixed for 15 minutes on an orbital shaker in order to achieve homogenisation.
From each well, three replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in a plate spectral photometer at 570 nm.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: percentage values of formazan production in comparison to the negative control

Basis:

mean

Time point:

other: 3 minutes

Score:

97.5

Reversibility:

not specified

Remarks on result:

other: test item

Results 2

Irritation parameter:

other: percentage values of formazan production in comparison to the negative control

Basis:

mean

Time point:

other: 30 minutes

Score:

95.3

Reversibility:

not specified

Remarks on result:

other: test item

Results 3

Irritation parameter:

other: percentage values of formazan production in comparison to the negative control

Basis:

mean

Time point:

other: 60 minutes

Score:

90.3

Reversibility:

not specified

Remarks on result:

other: test item

Results 4

Irritation parameter:

other: percentage values of formazan production in comparison to the negative control

Basis:

mean

Time point:

other: 15 minutes

Score:

57.6

Reversibility:

not specified

Remarks on result:

other: positive control

Results 5

Irritation parameter:

other: percentage values of formazan production in comparison to the negative control

Basis:

mean

Time point:

other: 45 minutes

Score:

23.6

Reversibility:

not specified

Remarks on result:

other: positive control

Any other information on results incl. tables

Mean Absorption Values:

Designation	Negative control	Positive control	Positive control	Test item	Test Item	Test item
Incubation time	60 min.	15 min	45 min.	3 min.	30 min.	60 min.
Mean – blank (Tissue 1)	1.544	0.875	0.356	1.596	1.586	1.393
Mean – blank (Tissue 2)	1.693	0.989	0.407	1.562	1.500	1.531
Mean of the two Tissues	1.619	0.932	0.382	1.579	1.543	1.462
Relative Standard Deviation of the two tissues	6.5%	8.6%	9.4%	1.5%	3.9%	6.7%

For the test item and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

% Formazan Production:

Designation	Positive control	Positive control	Test item	Test Item	Test item
Incubation time	15 min	45 min.	3 min.	30 min.	60 min.
% Formazan production (Tissue 1)	54.0%	22.0%	98.6%	98.0%	86.0%
% Formazan production (Tissue 2)	61.1%	25.1%	96.5%	92.6%	94.6%
% Formazan production Mean	57.6%	23.6%	97.5%	95.3%	90.3%

From the percentage Formazan production the following ET50 value was calculated:

Positive control:      21.68 minutes

For the test item Magnesium Ammonium Phosphate (MAP, "Berliner Pflanze"):, no value could be calculated as no relative absorption below 50 % was observed at any exposition time. ET50 is therefore stated as“> 60 minutes”.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item Magnesium Ammonium Phosphate (MAP, "Berliner Pflanze") is considered as not/minimal irritant in the Human Cornea Model test.

Executive summary:

This study was performed in order to evaluate the potential of Magnesium Ammonium Phosphate (MAP, "Berliner Pflanze") to evoke eye irritation in a human cornea model in an in-vitro study.

The eye irritation test refers to the production of irreversible tissue damage following the application of a test material on a human cornea model.

Irritating materials are identified by their ability to produce a decrease in cell viability as determined, by using the MTT reduction assay below defined threshold levels at specified exposure periods.

The principle of the human cornea model assay is based on the hypothesis, that irritating chemicals are able to penetrate the epithelium by diffusion or erosion, and are cytotoxic to the underlying cell layers.

The test itemMagnesium Ammonium Phosphate (MAP, "Berliner Pflanze") was applied to a three-dimensional human cornea model tissue for three different exposure times in duplicate (3 min., 30 min. and 60 min.).

100 mg of the solid test item were applied to each tissue.

Deionised water was used as negative control, 0.3 % Triton X 100 solution was used as positive control.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT which can be reduced to a blue formazan. Formazan production was measured by measuring the optical density (OD) of the resulting solution.

After treatment with the negative control, the absorbance values were well within the historical range and showed no cytotoxic effects. The positive control showed clear irritating effects for both treatment intervals.

After 3 minutes treatment with the test item, the relative absorbance values were reduced to 97.5 %. After 30 minutes treatment, relative absorbance values were reduced to 95.3 %. And after 60 minutes treatment, the relative absorbance values were reduced to 90.3 %.

 

From these values, the ET₅₀was derived. The ET₅₀of the test itemMagnesium Ammonium Phosphate (MAP, "Berliner Pflanze") is considered as “> 60 min” as after 60 minutes, absorption values of 90 % (compared with the negative control) were observed, lying far above 50 %.

 

Therefore, the test itemMagnesium Ammonium Phosphate (MAP, "Berliner Pflanze") is considered as

not/minimal irritant in the Human Cornea Model test.