Magnesium chloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2009 till february 2010

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:

equivalent or similar to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the testing of chemicals; Draft proposal for a new guideline, in vitro skin irritation: Reconstructed Human Epidermis (RhE) Test method,Version 4, 11 December 2009.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

magnesium chloride hexahydrate

IUPAC Name:

magnesium chloride hexahydrate

Constituent 2

Reference substance name:

7791-18-6

Cas Number:

7791-18-6

IUPAC Name:

7791-18-6

Details on test material:

water content (specification): 51-55 % (53.4 %)
Colour: colourless
Physical state: solid, crystals
Storage: at room temperature, in a tightly closed package
Solvent: water
Stability after opening: instable after repeated contact to air
pH: 5.5 - 7.0 (5 % solution at 20 °C)

Specific details on test material used for the study:

- Name of test material (as cited in study report):Magnesium chloride hexahydrate
- EC-Number: 232-094-6
- Physical state: Colourless; Solid, crystal
- Stability after opening: Instable after repeated contact to air
- Storage condition of test material: At room temperature, in a tightly closed package.
- pH: 5.5 - 7.0 (5% solution at 20 °C)
- Solvent: Water
No further information on the test material was stated.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (Skinethic). This skin model consists of normal (non-cancerous), adult human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period ans comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Vehicle:

water

Details on test system:

Preparation of the test Item: firstly, 5 µl of distilled water (A. dest.) were applied by a pipette to the epidermal surface in order to improve further contact between the test item and the epidermis. The water was gently spread with the pipette. Afterwards, the powder was applied to the epidermis surface.

Controls: controls were set up in parallel to the test item cultures in order to confirm the validity of the test. Negative control: sterile water and positive control: 5% sodium dodecyl sulfate.

Dose Groups. Negative control: 10 µL sterile water, Positive control: 10 µL 5% SDS solution, test Item: 10 mg + 5 µL A. dest. The test was performed on a total of 3 tissues per dose group.

Upon receipt of the EPISKIN-SMTM, the tissues were transferred into 12-well plates containing 2 mL prewarmed maintenance medium per well. The 12-well plates were incubated in a humidified incubator at 37 (+/-1) °C, 5.0% CO2 for at least 24 h. After this pre-incubation, the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the tissues were incubated at room temperature for 15 +/- 0.5 min. Afterwards, the tissues were washed with PBS to remove any residual test item. Excess PBS was removed by blotting bottom with blotting paper. The inserts were placed in a prepared 12-well plate containing 2 mL prewarmed fresh maintenance medium and post-incubated at 37 (+/- 1) °C, 5.0% CO2, humidified to 95% for 42 (+/-1) h.
After this incubation period, the insertes were transferred in a prepared 12-well plate containing 2 mL prewarmed MTT medium and further incubated for approximately 3 h at 37 (+/-1) °C, 5.0% CO2, humidified to 95%.
After the 3 h MTT incubation period, the tissues were placed on blotting paper to dry the tissues. Afterwards a total biopsy of the epidermis by using the special biopsy punch was performed and the epidermis was separated from the collagen matrix with the aid of forceps. Both parts (epidermis and collagen matrix) were transferred into suitable tubes and 500 µL of acidic isopropanol was added. Extraction was carried out protected from light over the weekend at 2 - 8°C.
At the end of the formazan extraction period the tubes were mixed by vortexing until solution colour became homogeneous.
If any visible cell/tissue fragments are in suspension, the tubes were centrifuged at 500 rpm to eliminate the fragments and avoid further possible interference with the absorbance readings.
Per each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 550 nm without reference wavelength in a plate spectrophotometer.
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with aqua dist.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

1. Negative control: 10 µL sterile water
2. Positive control: 10 µL 5% SDS solution
3. Test Item: 10 mg + 5 µL A. dest. Approximately 10 mg (26.3 mg/cm²) of the powder were applied to the epidermis surface.
The test was performed on a total of 3 tissues per dose group.

Duration of treatment / exposure:

15 +/- 0.5 min.

Duration of post-treatment incubation (if applicable):

42 h postincubation period and immediate determination of cytotoxic effects via MTT reduction assay

Test system

Vehicle:

water

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: Absorbance (550 nm)

Run / experiment:

15 min

Value:

ca. 0.831

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: other: mean. Time point: 15 mn. Max. score: 0.937. Reversibility: no data.

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min

Value:

112.9

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was =>50 % (112.9 %) after 15 min treatment and 42 h post incubation.Under the given conditions the test item showed no irritant effects

Any other information on results incl. tables

Pre-Experiment:

To check the MTT-reducing capability of the test item, 10 mg of the test item were mixed with 2 mL MTT medium and incubated for 3 h at 37±1 °C, 5.0% CO2.

Results after the treatment with MgCl2, 6H2O

Results of the test

Tissue Replicates	Negative Control			Positive Control			Test Item			Blank
	1	2	3	1	2	3	1	2	3	1	2	3
absolute OD 550-values	0.736	0.744	0.817	0.196	0.207	0.214	0.920	0.824	0.819	0.043	0.043	0.044
	0.759	0.764	0.856	0.207	0.208	0.227	0.980	0.864	0.839	0.043	0.044	0.044
netOD 550-values	0.693	0.701	0.774	0.153	0.164	0.171	0.877	0.781	0.776
	0.716	0.721	0.813	0.164	0.165	0.184	0.937	0.821	0.796
net mean of the duplicates	0.704	0.711	0.793	0.158	0.164	0.177	0.907	0.801	0.786
net mean of 3 replicate tissues	0.736*			0.166			0.831
Tissue viability[%]**	100.0	100.9	112.6	22.4	23.3	25.1	128.8	113.7	111.6
SD Tissue viability[%]***	7.0			1.4			9.4
mean relative tissue viability[%]	100.0			22.6			112.9

*   mean OD 550 nm of the three negative control tissues is >= 0.6

** mean relative tissue viability of the three positive control tissues is =< 30

*** the standard deviation (SD) obtained from the three concurrently tested tissues is =< 18%

To check the MTT-reducing capability of the test item 10 mg of the test item were mixed with 2 mL MTT medium and incubated for 3 h at 37±1 °C, 5.0% CO2. The mixture did not turn blue/purple, therefore the test item itself did not reduce MTT, the OD550 was 0.079 (blank: 0.084).

.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, MgCl2, 6H2O showed no skin irritant effects.

Executive summary:

The in vitro study according to the draft EC method B.46 (EpiSkinTM) was validated and considered of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, and for being used as a replacement of in vivo method OECD 404 for the purposes of distinguishing between R38 skin irritating and non-skin irritating test substances (ECVAM, 27.04.2007).

 

 The potential of the test item to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN- Standard Model™(Skinethic) comprising a reconstructed epidermis with a functional stratum corneum.

In the present study, Magnesium Chloride Hexahydrate was applied topically to the EPISKIN- SM™ tissue for 15 min followed by a 42 h postincubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with sterile water.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was =>50 % (112.9 %) after 15 min treatment and 42 h post incubation. The controls confirmed the validity of the study. The mean OD550 of the three negative control tissues was =>0.6. The mean relative tissue viability (% negative control) of the positive control was =< 30 % (22.6 %). The standard deviation of replicate tissues of all dose groups was <= 30 % (1.4 % - 9.4 %).