Magnesium zirconium oxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-03-02 to 2010-03-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline 440 (Part B: Methods for determination of toxicity and other health effects, Guideline B.40 BIS "In vitro corrosion: Human skin model test"

Deviations:

no

GLP compliance:

yes

Remarks:

Self-Certified

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium hydroxide

EC Number:

215-170-3

EC Name:

Magnesium hydroxide

Cas Number:

1309-42-8

Molecular formula:

H2MgO2

IUPAC Name:

magnesium dihydroxide

Details on test material:

Identification: Magnesium hydroxide
Molecular formula: Mg(OH)2
Molecular weight: 58.32
CAS Number: 1309-42-8
Stable under storage conditions: Stable

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

water

Details on test system:

The potential of magnesium hydroxide to induce skin corrosion was tested using a human three-dimensional epidermal model.
This model consisted of normal, human-derived epidermal keratinocytes which were cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Magnesium hydroxide was assessed for its ability to directly reduce MTT before the study was started to ensure that this would not interfere with the results obtained. Approximately 100 mg of magnesium hydroxide was added to a 24-well plate filled with 1 mL MTT medium. The mixture was incubated for 1 hr at room temperature in the dark. As a negative control, sterile Milli-Q water was tested concurrently.

On the day of receipt, skin tissues were stored on agarose at 4°C. The following day, at least 1 hour prior to starting the assay, the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of DMEM medium just reached the underside of the tissue. The plates were incubated for 1 hr 50 min at 37°C with 5% CO2. The medium was replaced with fresh DMEM medium and the tissue was moistened with 25 µL of Milli-Q water just prior to application of the test substance. The test was performed with a total of 4 tissues per test substance together with a negative control and positive control. 25 mg of Mg(OH)2 was applied directly on top of the moistened skin tissue. Two tissues were used for a 3-minute exposure to Mg(OH)2 and two for a 1-hour exposure. The negative and positive controls were treated with 50 µL Milli-Q water and 50 µL 8N KOH, respectively.

After the exposure period, the tissues were washed with PBS to remove residual test substance. Rinsed tissues were kept in 24-well plates on 300 µL DMEM until 6 tissues were dosed and rinsed.

To measure cell viability, the DMEM was replaced with 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 540 nm in triplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin corrosion potential of the test substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of magnesium hydroxide was applied directly on top of skin tissue which was moistened with 25 µL of Milli-Q water to ensure close contact to the tissue.

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

To measure cell viability, the DMEM was replaced with 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in 5% CO2.

Number of replicates:

4 tissues per test substance, 2 for 3 min exposure and 2 for 1 h exposure

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: % viability of skin cells

Value:

88

Remarks on result:

other: Basis: mean. Time point: 3 minutes. Max. score: 100.0.

Results 2

Irritation / corrosion parameter:

other: % viability of skin cells

Value:

95

Remarks on result:

other: Basis: mean. Time point: 1 hour. Max. score: 100.0.

In vivo

Irritant / corrosive response data:

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

Any other information on results incl. tables

Magnesium hydroxide was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that magnesium hydroxide did not interact with MTT.

The mean absorption at 540 nm measured after treatment with magnesium hydroxide and controls are presented in Table 1. Table 2 shows the mean tissue viability obtained after 3 minute and 1 hour treatments with magnesium hydroxide compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance.

A test substance is considered corrosive in the skin corrosion test if:

1. The relative mean tissue viability obtained after 3 minutes of treatment compared to the negative control tissues is decreased below 50%.

2. The relative tissue viability after 1 hour of treatment is decreased below 15%.

 

A test substance is considered to be non-corrosive if: 

1. The relative mean tissue viability obtained after 3 minutes of treatment compared to the negative control tissues is above 50%.

2. The relative tissue viability after 1 hour of treatment is not decreased below 15%. The relative mean tissue viability obtained after the 3 minute and 1 hour treatments with magnesium hydroxide compared to the negative control tissues were 88% and 95%, respectively. The absolute mean OD540 of the negative control tissues was within the historical control range. The mean relative tissue viability following 3 minutes and 1 hour of exposure to the positive control were 9%. Therefore, it was concluded that the test system was suitable for this analysis. Table 1: Mean absorption in the in vitro skin corrosion test with magnesium hydroxide (OD₅₄₀)

	3 minute application			1 hour application
	A	B	Mean ± SD	A	B	Mean ± SD
Negative control	1.683	1.699	1.691 ± 0.011	1.707	1.710	1.708 ± 0.002
Magnesium hydroxide	1.586	1.393	1.490 ± 0.137	1.664	1.578	1.621 ± 0.061
Positive control	0.150	0.143	0.147 ± 0.005	0.148	0.145	0.147 ± 0.002

  

Table 2: Mean tissue viability

 

	3 minute application viability (% of control)	1 hour application viability (% of control)
Negative control	100	100
Magnesium hydroxide	88	95
Positive control	9	9

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, it is concluded that magnesium hydroxide is not corrosive in the in vitro skin corrosion test.

Executive summary:

The potential of magnesium hydroxide to induce skin corrosion was tested using a human three-dimensional epidermal model. The possible corrosive potential of magnesium hydroxide was tested using topical application for either 3 minutes or 1 hour. Twenty five mg of magnesium hydroxide was added directly on top of the skin tissue which was moistened with water. Water and potassium hydroxide were used as the negative and positive control substances, respectively.

The positive control had a mean relative tissue viability of 9% after 3 minutes exposure, and the absolute mean optical density of the negative control tissues was within the historical control range, indicating the acceptability of the assay.

The mean relative tissue viabilities for magnesium hydroxide after 3 minute and 1 hour treatments were 88% and 95%, respectively. Because the mean relative tissue viability for magnesium hydroxide was not below 50% after the 3 minute treatment or 15% after the 1 hour treatment, it was concluded that magnesium hydroxide is not corrosive under the conditions of this test.