Amorphous Mesoporous Magnesium Carbonate

Endpoint summary

Description of key information

Upsalite is not considered to be irritating to the skin and main constituents are not considered to be irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08/05/2018-11/05/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Reconstructed Human Epidermis Test Method - adopted in 2013
Method followed: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) with Reconstructed Human
Epidermal Model

GLP compliance:

yes

Specific details on test material used for the study:

Q0002-1701

Test system:

other: reconstructed Human Epidermal Model.

Vehicle:

not specified

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 85.2

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

According to the EU and GHS classification (R38/Category 2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below 50% of the mean viability of the negative controls.

Interpretation of results:

GHS criteria not met

Conclusions:

According to results obtained from this test implementation, the viability of Reconstructed human epidermal model is > 50%. The product: UPSALITE Q0002-1701 is classified as: NI (Not Irritant)/not required classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation, other

Remarks:

ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15.3.2015-16.3.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to Guidelines in a GLP-certified laboratory

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Remarks:

Self-certified

Specific details on test material used for the study:

Identification: Magnesium hydroxide
Molecular formula: Mg(OH)2
Molecular weight: 58.32

Species:

other: Bovine

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. The eyes were collected and transported in physiological saline in a suitable container and were used within 30 minutes.

Vehicle:

physiological saline

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Unknown

Duration of treatment / exposure:

topical treatment for 240 ± 10 minutes

Observation period (in vivo):

After incubation with the test substance, the solutions were removed, fresh cMEM was added and an opacitiy determination was performed without any further incubation

Number of animals or in vitro replicates:

Three corneas were used in each treatment group

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: After the incubation with the test substance, corneas were washed at least 3 times with cMEM prior to determination of opacity
- Time after start of exposure: 240 ± 10 minutes

OPACITY:
The opacitometer determined the difference in light transmission between each control and treated cornea and an air filled chamber. The numerical opacity value was displayed and recorded. The change in opacity for each individual cornea was calculated by subtracting the initial opacity reading from the post-treatment reading. The corrected opacity was calculated by subtracting the opacity of the negative control.

PERMEABILITY
Following the final opacity method, permeability of the cornea to Na-fluorescein was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of Na-fluorescein solution. The corneas were completely covered and were incubated in a horizontal position for 90 ± 5 minutes at 37 ± 1 °C.

After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm of each sampling tube was measured in triplicate using a microplate reader.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 240 min

Score:

ca. 5.1

Max. score:

7.1

Remarks on result:

other: in vitro irritancy score

Irritation parameter:

other: Opacity

Basis:

mean

Time point:

other: 240 min

Score:

ca. 5

Max. score:

7

Irritant / corrosive response data:

See " Any other information on results incl. tables".

Other effects:

ND

Table 1 Summary of opacity, permeability and in vitro scores

Treatment	Mean Opactiy	Mean Permeability	MeanIn vitroIrritation Score^1,2
Negative control	0	0.000	0.0
Positive control	80	2.490	117
Magnesium hydroxide	5	0.006	5.1

1. Calculated using the negative control mean opacity an mean permeability values.

2. In vitro irritancy score (IVIS)= mean opacity + (15X mean OD₄₉₀value).

Table 2. Opacity score

Eye	Opacity before treatment	Opacity after treatment	Final opacity	Negative control corrected final opacity	Mean opacity
Negative control
1	0	0	0	0	0
2	0	1	1	1
3	0	0	0	0
Positive control
4	0	80	80	80	80
5	0	79	79	79
6	0	81	81	81
Magnesium hydroxide
10	0	6	6	6	5
11	0	3	3	3
12	0	7	7	7

Table 3. Permeability score (corrected)

e	Dilution factor	Corrected OD4901	Corrected OD4902	Corrected OD4903	Average OD490	Final OD490	Average OD
Negative control
1	1	0.014	0.001	0.004	0.006	0.006	0.000
2	1	0.000	0.000	0.003	0.001	0.001
3	1	-0.001	-0.008	-0.009	-0.006	-0.006
Positive control
4	6	0.369	0.366	0.372	0.369	2.214	2.490
5	6	0.349	0.362	0.354	0.355	2.130
6	6	0.527	0.518	0.519	0.521	3.126
Magnesium hydroxide
10	1	0.008	0.008	0.011	0.009	0.009	0.006
11	1	0.008	0.002	0.000	0.003	0.003
12	1	0.006	0.004	0.005	0.005	0.005

Table 4. In vitro irritancy score

Eye	Negative control corrected final opacity	Negative control corrected final OD490	In vitro irritancy score
Negative control
1	0	0.006	0.1
2	0	0.001	1.0
3	0	-0.006	-0.1
Positive control
4	80	2.214	113.2
5	79	2.130	111.0
6	81	3.126	127.9
Magnesium hydroxide
10	6	0.009	6.1
11	3	0.003	3.0
12	7	0.005	7.1

In vitro irritancy score (IVIS) = mean opacity + (15 x mean OD₄₉₀value)

Interpretation of results:

GHS criteria not met

Remarks:

Not irritating

Conclusions:

Since the mean in vitro irritancy score for Magnesium Hydroxide was below 55.1 after 240 minutes treatment, Magnesium Hydroxide is not classed as an irritant in the BCOP test.

Executive summary:

Screening for the eye irritancy potential of Magnesium hydroxide using the BCOP test. The study procedures were based on OECD guidelines. Magnesium hydroxide did not induce ocular irritation through both endpoints, resulting in a meanin vitroirritancy score of 5.1 after 240 minutes of treatment. Since this score is below 55.1 after 240 minutes treatment, Magnesium hydroxide is not classified as an irritant in the BCOP test.

Reference 2

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Instead of using fresh bovine corneas, four laboratories (laboratories 2, 3, 8 and 10), agreed to perform the experiments with preserved corneas. It was of particular interest to know whether both tissues would respond similarly to chemical injury. In some locations, it was sometimes difficult to get the eyes early enough in the morning to run a long experiment with solid substances (4-hr incubation). L'Orral has routinely used both fresh and preserved corneas and similar results were obtained (M. Cottin, personal communication, 1991). The protocol used to preserve corneas was the following. Corneas were dissected as usual with a 2-3-mm scleral rim. They were then washed three times, each for 15 min, in HBSS supplemented with antibiotics (penicillin/streptomycin, 100 U/100/ag per ml). After rinsing in normal HBSS, they were placed individually into wells of six-well culture plates, each of them containing 12 ml preservative medium. The latter consisted of M 199 medium (Sigma M-3769) containing 5% (w/v) dextran (Sigma D-1662; mol. wt 40,000), adjusted to pH 7.4, and prepared daily. Plates were then placed in the refrigerator at 4-5°C until the next day. For use, the plates were removed from the refrigerator and left on the bench for 30 min at room temperature and the corneas were mounted in holders and filled with MEM. The next steps were identical to those used for fresh corneas

GLP compliance:

not specified

Specific details on test material used for the study:

Test material is from Aldrich

Species:

other: Bovine

Details on test animals or tissues and environmental conditions:

Bovine eyes were collected from a commercial abattoir in a plastic jar containing 1 litre HBSS for approximately 25 eyes.Buffer storage and eyes transportation to the laboratorieswere performed at ambient temperature, and the eyes were used within 2 hr of the killing of the animals. During dissection great care was taken to avoid damage of corneal surfaces (epithelial and endothelial). Each cornea was carefully examined,and those presenting defects, such as neovascularization, pigmentation, opacity or scratches were discarded.Globes were first dissected free of surrounding tissues and placed in a jar containing fresh HBSS. Selected corneas were dissected with a 2-3-mm rim of sclera for easier handling, and stored in a petri dish containing HBSS until use. Corneas were then mounted in holders filled with MEM, and incubated for 1 hr in a water-bath at 32°C.

Vehicle:

other: MEM; minimum essential medium

Controls:

yes, concurrent vehicle

Amount / concentration applied:

Solids were prepared as an approximate 20% solution or suspension (200mg+lml) in MEM using a mortar and pestle for homogeneous preparations, and a volume of 0.75 ml was applied on the epithelium with an appropriate syringe and needle.

Duration of treatment / exposure:

Solid test substances are incubated at 32 ± 1°C for 240 ± 10 minutes. The compound was then removed and the epithelium was washed at least three times (until thecornea was free of particles; gentle swirling movementsof the holders were sometimes necessary) with approximately 4ml medium.

Duration of post- treatment incubation (in vitro):

Both compartments were refilled with medium, and opacity measurement was performed immediately without any further incubation.

Number of animals or in vitro replicates:

Table summarizes the results obtained for test substances by the 11 laboratories. Each value represents the mean in vitro score from six corneas.
A mean in vitro score from all laboratories was also calculated.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS :
Bovine eyes are typically obtained from a local cattle abattoir, where the eyes are excised as soon as possible after sacrifice. Care should be taken to avoid damaging the cornea during the enucleation procedure. Eyes are collected in a suitable container in which they are immersed in HBSS containing the antibiotics penicillin (100 IU/mL) and streptomycin (100 μg/mL) .The eyes are used within five hours of sacrifice.

QUALITY CHECK OF THE ISOLATED CORNEAS :
Carefully is examined all eyes macroscopically. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation, and neovascularization are rejected.Carefully remove the cornea from each selected eye by making an incision with a scalpel 2 to 3 mm outside the cornea, then by cutting around the cornea with dissection scissors, leaving a rim of sclera to facilitate handling. Carefully peel off the iris and lens, ensuring no fragments of these tissues are remaining on the cornea. Take care to avoid damaging the corneal epithelium and endothelium during dissection. Store the isolated corneas in a petri dish containing HBSS until they are mounted in holders. Examine the corneas before use, and discard those with defects. Mount the corneas in holders (one cornea per holder) by placing the endothelial side of the cornea against the O-ring of the posterior chamber. Place the anterior chamber over the cornea and join the chambers together by tightening the chamber screws. Care should be taken not to shift the two chambers to avoid damaging the cornea. Fill both chambers with fresh complete MEM (about 5 mL), always filling the posterior chamber first to return the cornea to its natural curvature. Care should be taken when adding or removing liquid from the posterior chamber to avoid the formation of bubbles and to minimize shear forces on the corneal endothelium. Seal each chamber with plugs provided with the holders. Incubate the holders in a vertical position at 32 ± 1°C for at least 60 minutes. At the end of the initial 1-hour incubation period, examine each cornea for defects, such as tears or wrinkling. Discard corneas with any observed defects.


NUMBER OF REPLICATES : minimum of three corneas

SOLVENT CONTROL USED (if applicable)


APPLICATION DOSE AND EXPOSURE TIME

TREATMENT METHOD: Solid nonsurfactant test substances:Solid test substances are tested on the cornea as a 20% (w/v) solution or suspension prepared in an appropriate solvent/vehicle.
Solid test substances are incubated at 32 ± 1°C for 240 ± 10 minutes.

POST-INCUBATION PERIOD: no, Thus, immediately following the rinsing process, both chambers are refilled (posterior chamber first) with fresh complete MEM, and the post-treatment opacity readings are taken. During the post-treatment opacity reading, visual observations are performed for each cornea and, if necessary, are recorded in the workbook.


METHODS FOR MEASURED ENDPOINTS:
In the BCOP test method, changes in corneal opacity caused by chemical damage are determined by measuring decreases in light transmission through the cornea. Changes in permeability of the cornea resulting from chemical damage are determined by measuring increases in the quantity of sodium fluorescein dye that passes through all corneal cell layers.
Opacity measurement. The opacitometer (Electro-Design, Riom, France) determines the difference inlight transmission between a treated and a control cornea, and displays a numerical opacity value (arbitrary units). The apparatus was previously calibrated with standardized opaque sheets of polyester, and values obtained with test substances generally ranged between 0 and 150.
Permeability. This second step of the assay wasperformed immediately after the measurement of opacity. The medium was again removed from both chambers of holders, and replaced by fresh medium in the posterior compartment, and by 1 ml fluorescein solution (0.4% for liquids and surfactants, 0.5% for solids) in contact with the epithelium. Corneas were then incubated in a horizontal position for 90 min at 32°C. Medium from the posterior chamber was then removed, and its optical density (O.D.) determined with a spectrophotometer at 490 nm.

For each substance evaluated, the mean value of opacity + SD and the mean value of permeability (expressed as O.D. units + SD) were calculated. An in vitro score for irritancy combining both parameters was also
calculated using the following formula:In vitro score = opacity value + 15 times O.D. valueScoring: Alternatively, compounds can be classified more simply as irritating (moderate plus severe, i.e. score ~>25.1) or non-irritating (mild, i.e. score ~<25.0),

Irritation parameter:

cornea opacity score

Run / experiment:

6

Value:

ca. 3

Vehicle controls validity:

not specified

Irritation parameter:

cornea opacity score

Run / experiment:

6

Value:

ca. 6

Vehicle controls validity:

not specified

Irritation parameter:

cornea opacity score

Run / experiment:

6

Value:

ca. 0

Vehicle controls validity:

not specified

Irritation parameter:

cornea opacity score

Run / experiment:

6

Value:

ca. 1

Vehicle controls validity:

not specified

Irritation parameter:

cornea opacity score

Run / experiment:

6

Value:

ca. 7

Vehicle controls validity:

not specified

Interpretation of results:

GHS criteria not met

Conclusions:

Mean score 3, reversibility 3 days.
Agreement between laboratories when data are grouped in two classes (non-irritant, score < 25.0; irritant score /> 25.1). The results of all laboratories ; non-classified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

In thein vitro skin irritation (upsalite )and the in vivo eye irritation studies, magnesium carbonate and magnesium hydroxide were non-irritant and therefore requires no classification under the CLP.