Magnesium chloride

Endpoint summary

Description of key information

Irritation/corrosion studies have been conducted with magnesium chloride hexahydrate.

The results can be extrapolated to magnesium chloride anhydrous.

Thus:

- For skin irritation, the MgCl2 is not irritating on skin (EU method B46).

- For eye irritation, the MgCl2 is not irritating on eyes (OECD 405)

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:

October 2009 till february 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

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

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

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.

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

Vehicle:

water

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.

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

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).

.

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 %).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2009 till March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Magnesium chloride hexahydrate
- Physical state: Colourless; Solid, crystals
- 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.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Species/strain: Healthy New Zealand White Rabbits, Crl: KBL (NZW)
Source: Charles River Deutschland, D-97633 Sulzfeld
Sex: female
Body weight at the beginning of the study: > 2kg
Age at the beginning of the study: approximately 21 weeks old
The animals were derived from a controlled full barrier maintained breeding system (SPF).

Housing and Feeding Conditions: semi-barrier in an air-conditioned room, temperature: 18 (+/-3)°C, relative humidity: 55 (+/-10)%, artificial light, sequence being 12 hours light, 12 hours dark, air change: at least 10 x / hour, free access to autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre, free access to water, housed in ABS - plastic rabbit cages, floor 4200 cm2 and adequate acclimatisation period (at least 5 days).

Vehicle:

unchanged (no vehicle)

Controls:

other: The untreated contralateral eye served as control.

Amount / concentration applied:

A dose of 0.1 g of the test item was applied to the test site.

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

Observation at 1, 24, 48, and 72 hours and 4 to 6 days

Number of animals or in vitro replicates:

3 female rabbits

Details on study design:

Approximately 24 hours before the test and immediately prior to the application both eyes of each animal were examined. A health inspection was performed to ensure the good state of health of the animals. None of the animals showed eye irritation, ocular defects, or pre-existing corneal injury.

The test item was applied at a single dose in the conjunctival sac of one eye of each test animal after pulling the lower lid away from the eyeball. The lids were then gently held together for about 1 second in order to prevent loss of the material. 72 hours post-instillation as well as at the end of the prolonged observation period the treated eyes were examined with the aid of a fluorescein solution

The eyes were examined for signs of irritation throughout the observation period. Individual reactions of each animal were recorded at each time of observation. The observations were recorded by a toxicologist and confirmed by another. Nature, severity and duration of all lesions observed were described. For the calculation only the 24-, 48- and 72-hour readings were used.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

0

Remarks on result:

other: Each animal showed no cornea opacity at any time

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

0

Remarks on result:

other: Each animal showed no iris effect at any time

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 1.67

Max. score:

2

Reversibility:

fully reversible within: 6 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0.67

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 1.33

Max. score:

2

Reversibility:

fully reversible within: 4 days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 1

Max. score:

2

Reversibility:

fully reversible within: 4 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0.67

Max. score:

2

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0.33

Max. score:

2

Reversibility:

fully reversible within: 48 hours

Other effects:

There were no significant body weight changes, no individual systemic and local findings during the observation period.

Eye Irritation Scores – Animal No. 1

 

	Single data				Average score (24, 48 and 72 hours)
	Time post application
	1 hour	24 hours	48 hours	72 hours
	T/C	T/C	T/C	T/C
Cornea	0/0	0/0	0/0	0/0	0
Iris	0/0	0/0	0/0	0/0	0
Conjunctival redness	2/0	2/0	2/0	1/0	1.67
Conjunctival chemosis	3/0	1/0	1/0	0/0	0.67

 

	Single data
	Time post application
	4 days	5 days	6 days	7 days	8 days
	T/C	T/C	T/C	T/C	T/C
Cornea	0/0	0/0	0/0	-	-
Iris	0/0	0/0	0/0	-	-
Conjunctival redness	1/0	1/0	0/0	-	-
Conjunctival chemosis	0/0	0/0	0/0	-	-

 

Eye Irritation Scores – Animal No. 2

 

	Single data				Average score (24, 48 and 72 hours)
	Time post application
	1 hour	24 hours	48 hours	72 hours
	T/C	T/C	T/C	T/C
Cornea	0/0	0/0	0/0	0/0	0
Iris	0/0	0/0	0/0	0/0	0
Conjunctival redness	2/0	2/0	1/0	1/0	1.33
Conjunctival chemosis	2/0	1/0	1/0	1/0	1.0

 

	Single data
	Time post application
	4 days	5 days	6 days	7 days	8 days
	T/C	T/C	T/C	T/C	T/C
Cornea	0/0	-	-	-	-
Iris	0/0	-	-	-	-
Conjunctival redness	0/0	-	-	-	-
Conjunctival chemosis	0/0	-	-	-	-

Eye Irritation Scores – Animal No.3

 

	Single data				Average score (24, 48 and 72 hours)
	Time post application
	1 hour	24 hours	48 hours	72 hours
	T/C	T/C	T/C	T/C
Cornea	0/0	0/0	0/0	0/0	0
Iris	0/0	0/0	0/0	0/0	0
Conjunctival redness	2/0	2/0	0/0	0/0	0.67
Conjunctival chemosis	2/0	1/0	0/0	0/0	0.33

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, MgCl2, 6H2O is not irritating to eye.

Executive summary:

Under the conditions of this study, single ocular instillation of the test item Magnesium chloride hexahydrate to rabbits at a dose of 0.1 g produced irritant effects, which were fully reversible within 6, 4 and 2 days post-instillation in animal no. 1, 2 and 3, respectively.

Neither mortalities nor significant clinical signs of toxicity were observed.

According to the GHS criteria, the MgCl2, 6H20 is not irritating to eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Irritation/corrosion studies have been conducted with magnesium chloride hexahydrate.

Skin irritation / corrosion.

Two studies indicate no skin irritant effects for MgCl2, 6H20. The study of Stuhlmann D., 2010 (reliability 1) is selected as key study. In this 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 (EU method B46). Irritant potential was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with sterile water. MgCl2, 6H2O showed no irritant effect

Eye irritation

Two studies indicate no eye irritant effects for MgCl2, 6H20. The study of Ahuja V., 2010 (reliability 1) is selected as key study. Under the conditions of this study (OECD 405), single ocular instillation of the test item Magnesium chloride hexahydrate to New Zealand white rabbits at a dose of 0.1 g produced irritant effects, which were fully reversible. MgCl2, 6H2O is not irritating on eyes.

Effect level: empty Endpoint conclusion: Adverse effect observed

Justification for classification or non-classification

On the basis of study results, the MgCl2 was not classified for skin and eye irritation under the CLP regulation 1272/2008.