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Description of key information

Key studies demonstrate a lack of irritation/corrosion potential: 
Data are read-across from a structural analogue, i.e. magnesium chloride hexahydrate.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
other: in vitro
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2010-01-19 to 2010-01-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study reliable with restrictions Minor deviation without an effect on the results: - The historical acceptance range was not given.
Qualifier:
equivalent or similar to guideline
Guideline:
other: Commision regulation (EC) No. 440/2008 B.46
Deviations:
no
Qualifier:
equivalent or similar 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:
equivalent or similar 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 (incl. QA statement)
Remarks:
signed 2009-04-06
Details on test animals or test system and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.
Vehicle:
water
Remarks:
(distilled water (A. dest.))
Amount / concentration applied:
TEST MATERIAL AND VEHICLE
- Amount(s) applied (volume or weight with unit): Firstly, 5µl 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 approximately 10 mg (26.3 mg/cm^2) of powder were applied to the epidermis surface.
No further information on the amount/concentration applied was stated.
Duration of treatment / exposure:
15 +/- 0.5 min.
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
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 (Delta Select, Lot No. 12903C)
Positive control: 5% sodium dodecyl sulfate (SDS; AppliChem, Art.-No.A1112,0500, CAS No.: 151-21-3, Lot No. 8T009926) in A. dest.

DOSE GROUPS:
1. Negative control: 10 µL sterile water
2. Positive control: 10 µL 5 % SDS solution
3. Test item: 10 mg +/- 5 µL A. dest.
The test was performed on a total of 3 tissues per dose group.

TEST SYSTEM:
The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SMTM (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 comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

MTT SOLUTION:
- MTT stock solution: 3 mg/mL MTT in PBS
- MTT medium: MTT stock solution will be diluted 1 + 9 with assay medium (final concentration 0.3 mg/mL). The solution was kept at -20 °C for not longer than 1 year and was not thawn more than 10 times.

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 1 h at room temperature. If the mixture turns blue/purple, the test item is presumed to have reduced MTT.

EXPERIMENTAL PROCEDURE:
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 +/- 1h.
After this incubation period the inserts were transferred in a prepared 12-well plate containing 2 mL prewarmed MTT medium and further incubated for approximately 3 h +/- 5 min. 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 mytrix with the aid of forceps. Both parts (epidermis and collagen matrix) were transferred into suitable tubes and 500 µL of acidic isopropanol (0.04 N HCl in 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.

DATA ANALYSIS:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with aqua dest. The test item is considered to be irritating to skin (R38), if the tissue viability after 15 min of exposure and 42 h of post-incubation is less or equal to 50%.

TEST ACCEPTANCE CRITERIA:
The test meets acceptance criteria if:
- mean OD550nm 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%
No further information on the study design was stated.
Irritation parameter:
other: relative tissue viability (%)
Basis:
mean
Time point:
other: after 15 min incubation
Score:
112.9
Reversibility:
no data
Remarks on result:
other: OD550 nm
Irritant / corrosive response data:
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.

PRE-EXPERIMENT:

The mixture did not turn blue/purple, therefore the test item itself did not reduce MTT. The OD550 was 0.079 (blank: 0.084).

EXPERIMENT:

Tissue Replicates

    Negative control

 1           2          3

     Positive control

   1          2           3

          Test item

   1           2           3

            Blank

  1           2          3

Absolute OD550-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

net OD550-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 duplicats

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 viablility [%]

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 OD550nm 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%

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 maximum inter tissue difference of replicate tissues of all dose groups was ≤30% (1.4 % - 9.4 %).

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In this study under the given conditions the test item showed no irritant effects. The test item is classified as "non irritant (NI)".
The test item should not be classified and labeled as skin irritant according to Directive 67/548/EEC.
The test item should not be classified and labeled as skin irritant according to regulation (EC) No.: 1272/2008.
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). Hence, an additional endpoint study record for waiving the in vivo test is not provided.

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2010-02-03 to 2010-02-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
, 2002-04-24
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2009-04-06
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS (The animals were derived from a controlled full barrier maintained breeding system (SPF). According to Art. 9.2, No.7 of the German Act on Animal Welfare the animals were bred for experimental purposes.)
- Source: Charles River Deutschland, D-97633 Sulzfeld, Germany
- Age at study initiation: approximately 21 weeks old
- Weight at study initiation: Animal No. 1: 4.1 kg; Animal No. 2: 4.0 kg; Animal No. 3: 3.9 kg
- Housing: Semi-barrier in an air-conditioned room; Housed in ABS - plastic rabbit cages, floor 4200 cm2
- Diet: Free access to autoclaved hay and to Altromin 2123 maintenance diet for rabbits (lot no. 1208), rich in crude fibre
- Water: Free access to tap water (drinking water, municipal residue control, microbiol. controlled periodically)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 +/- 3°C
- Relative humidity: 55 +/- 10%
- Air changes: At least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A dose of 0.1 g of the test item was applied to the test site. 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. The untreated contralateral eye served as control.
No further information on the amount/concentration applied was stated.
Duration of treatment / exposure:
No data
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:
The in vivo test was performed initially using one animal. The results of the initial test did not indicate the test item to be corrosive or a severe irritant to the eye using the procedure described. In order to confirm the response, two additional animals were treated in the same manner.

PREPARATION OF THE ANIMALS.
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.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The eyes were not rinsed.

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: 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 (Fluoreszein SE Thilo, lot no. H 901, expiry date: 12/2010).

-OTHER: The eyes were examined for signs of irritation throughout the observation period. Nature, severity and duration of all lesions observed were described.
No further information on the study design was stated.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24, 48, and 72 hours
Score:
1.67
Max. score:
3
Reversibility:
fully reversible within: 6 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24, 48, and 72 hours
Score:
0.67
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24, 48, and 72 hours
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 4 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24, 48, and 72 hours
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 4 days
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24, 48, and 72 hours
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24, 48, and 72 hours
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
The test item produced irritant but reversible ocular effects after instillation into the eyes of 3 female rabbits.
Upon fluorescein examinations at the end of the observation period of 72 h and further at the end or prolonged observation period no corneal lesions were found in any animal.
Conjunctival discharge was observed in all animals, 1 hr post-instillation. No conjunctival discharge was observed 2 days post-instillation in all the animals.
Conjunctival redness and chemosis were also observed in all animals.

Other effects:
Neither mortalities nor significant clinical signs of toxicity were observed. There were no significant body weight changes during observation period.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the present 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. Neither mortalities nor significant clinical signs of toxicity were observed.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments on the approximation of the laws, regulations and administrative provision relating to the classification, packaging and labelling of dangerous substances and the results obtained under the present test conditions magnesium chloride hexahydrate was non-irritating to eyes, hence, no labelling is required.
According to the EC Regulation 1272/2008 and subsequent regulations, the test item is non-irritating to eyes; no classification and labelling of the substance is necessary.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Read across from MgCl2to Mg:

The effect of acute toxicity of magnesium chloridehexahydrate and magnesium is based on the concentration of dissolved Mg2+and therefore on the solubility of each substance.The water solubility of magnesium (6.7 mg/L at 21°C/pH 10.8) is 106orders of magnitude lower than the water solubility of magnesium chloride hexahydrate (1670 g/L at 20°C). Thus, read across to magnesium, based on worst case consideration, is justified.

Discussion:

Skin irritation:

Reference Stuhlmann & Lehmeier (2010) is considered as the key studies for in vitro skin irritation and will be used for classification. The overall irritation result is as follows:

Relative viability: mean after 15 min incubation 113% (max. score: 129%); evaluated by MTT reduction.

 

The classification criteria according to regulation (EC) 1272/2008 as irritating to skin are not met since the mean rel. viability is >50% of the control, hence no classification required. 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). Hence, an additional endpoint study record for waiving the in vivo test is not provided.

 

 

Eye irritation:

Reference Ahuja (2010) is considered as the key study for eye irritation and will be used for classification. The overall irritation results are as follows:

Corneal opacity, iritis, chemosis and conjunctivae, 24, 48 and 72h after application: max score=2; effects were reversible within max 6 days

The classification criteria according to regulation (EC) 1272/2008 as irritating to eyes are not met. Hence, no classification and labelling are required according to regulation (EC) 1272/2008.

 

Respiratory irritation:

The classification as respiratory irritant is covered under the endpoint specific target organ toxicity- single exposure and repeated exposure. Please refer to the endpoint summaries on acute toxicity (endpoint 7.2) and repeated dose toxicity (endpoint 7.5) for further information.


Justification for selection of skin irritation / corrosion endpoint:
Read-across information from a GLP study with magnesium chloride hexahydrate as test item.

Justification for selection of eye irritation endpoint:
Read-across information from a GLP guideline study with magnesium chloride hexahydrate as test item.

Justification for classification or non-classification

According to the data presented in this section no classification and labelling is required for magnesium according to Regulation (EC) 1272/2008 and subsequent adaptations.