Registration Dossier

Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Reference substance name:
Magnesium chloride
EC Number:
232-094-6
EC Name:
Magnesium chloride
Cas Number:
7786-30-3
IUPAC Name:
magnesium dichloride
Constituent 2
Reference substance name:
7791-18-6
Cas Number:
7791-18-6
IUPAC Name:
7791-18-6
Constituent 3
Reference substance name:
magnesium chloride hexahydrate
IUPAC Name:
magnesium chloride hexahydrate
Test material form:
solid: crystalline
Details on test material:
- 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 animals

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.

Test system

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.

Results and discussion

In vivo

Results
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.

Any other information on results incl. tables

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 %).

Applicant's summary and conclusion

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.