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Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation / corrosion, other
Remarks:
other: validated "in vitro" test method
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-02-07 to 2012-02-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: EpiSkin™ corrosivity assay (Invittox 118)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 431
Qualifier:
equivalent or similar to guideline
Guideline:
other: EU test method B.40 bis
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of tri-µ-(2-ethylhexanoato-O)-bis(N,N',N''-trimethyl-1,4,7-triazacyclononane-N,N',N'')dimanganese and µ-(acetato-O)-di-µ-(2-ethylhexanoato-O)-bis(N,N',N''-trimethyl-1,4,7-triazacyclononane-N,N',N'')dimanganese
EC Number:
700-806-9
Molecular formula:
C42H87Mn2N6O6 and C36H75Mn2N6O6
IUPAC Name:
Reaction mass of tri-µ-(2-ethylhexanoato-O)-bis(N,N',N''-trimethyl-1,4,7-triazacyclononane-N,N',N'')dimanganese and µ-(acetato-O)-di-µ-(2-ethylhexanoato-O)-bis(N,N',N''-trimethyl-1,4,7-triazacyclononane-N,N',N'')dimanganese
Constituent 2
Reference substance name:
Reaction mass of tri- µ -(2-ethylhexanoato-O)-bis(N,N',N''-trimethyl-1,4,7-triazacyclononane-N,N',N'')dimanganese and µ -(acetato-O)-di- µ -(2-ethylhexanoato-O)-bis(N,N',N''-trimethyl-1,4,7-triazacyclononane-N,N',N'')dimanganese
IUPAC Name:
Reaction mass of tri- µ -(2-ethylhexanoato-O)-bis(N,N',N''-trimethyl-1,4,7-triazacyclononane-N,N',N'')dimanganese and µ -(acetato-O)-di- µ -(2-ethylhexanoato-O)-bis(N,N',N''-trimethyl-1,4,7-triazacyclononane-N,N',N'')dimanganese
Details on test material:
- Name of test material (as cited in study report): Manganese Octoate/Acetate - MeTACN complex
- Physical state: white solid
- Storage condition of test material: room temperature in the dark under nitrogen
- Lot/batch No.: 16D16603

Test animals

Details on test animals or test system and environmental conditions:
Not applicable - this is an in vitro study and no animals were used

Test system

Vehicle:
water
Controls:
not required
Amount / concentration applied:
20 mg of the solid test item was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. 100 ul of 0.9% w/v sodium chloride solution was added for wetting of the test item. Duplicate tissues, treated with 50 ul of 0.9% wlv sodium chloride solution served as negative
controls. Duplicate tissues, treated with 50 ul of glacial acetic acid served as positive controls.
Duration of treatment / exposure:
Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes.
Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes.
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
Episkin™ model kit 0.38cm2 was supplied by Skinethic, Nice, France on 2012-02-07

EXPOSURE:
2.2 ml of assay medium, warmed to approximately 37°C, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column.

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes. 20 mg of the solid test item was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. 100 ul of 0.9% w/v sodium chloride solution was added for wetting of the test item. Duplicate tissues, treated with 50 ul of 0.9% wlv sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 ul of glacial acetic acid served as positive controls. The treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.

REMOVAL OF TEST SUBSTANCE:
At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item. Each rinsed tissue was placed into the third column of the 12—welI plate until all tissues were rinsed.

MTT ASSAY:
2.2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12 well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 hours +/- 5 minutes at room temperature in a biological safety cabinet ensuring that the plates were protected from light. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was taken using the Episkin™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 ml micro tubes containing 850 ul of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MITT—loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 ul samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 ul of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240-minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with 0.9% w/v sodium chloride solution. The relative mean viabilities were calculated in the following way: Relative mean viability (%) = (mean ODM540 of test item)/(mean ODM540 of negative control) x 100
Classification of corrosivity potential was based on relative viabilities for each exposure time according to the following prediction model:

INTERPRETATION OF RESULTS:
The mean OD value obtained for the duplicate tissues per test item were used to calculate the percent viability relative to the negative control, which was arbitrarily set at 100%.
According to EU CLP Cat.1 and DSD (67/548/EEC):
The test item is considered to be corrosive to skin:
(1) if the viability after 3 minutes exposure is less than 50%, or
(2) if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%.
The test item is considered to be non-corrosive to skin:
(1) if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

ACCEPTABILITY OF THE ASSAY:
The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability met the acceptance criterion if the mean OD570 of the two tissues in both treatment intervals was ≥ 0.8.
The assay met the acceptance criterion if mean relative tissue viability of the positive control was ≤ 30%.
No further information on the study design was stated.



SCORING SYSTEM:

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: relative viability (%)
Basis:
mean
Time point:
other: after 3 mins exposure
Score:
85
Irritation parameter:
other: relative viability (%)
Basis:
mean
Time point:
other: after 60 mins exposure
Score:
61.9
Irritation parameter:
other: relative viability (%)
Basis:
mean
Time point:
other: after 240 mins exposure
Score:
80.4
Irritant / corrosive response data:
After exposure to the test item cobalt oxyhydroxide the relative absorbance values were unaltered at 106.9% after 3 minutes. After the 1 hour exposure relative absorbance values were reduced to 80.7%. Both values did not exceed the threshold for corrosivity of 50% for the 3 minutes exposure and 15% for the 1 hour exposure. Therefore, the test item was not considered to be corrosive.

Any other information on results incl. tables

Results

Item  Exposure Period  Mean OD540of
duplicate tissues
 Relative mean
viability (%)
Negative Control Item 240 Minutes 0.260 100*
Positive Control Item 240 Minutes 0.015 5.8
Test Item 240 Minutes 0.209 80.4
60 Minutes 0.161 61.9
3 Minutes 0.221 85.0
* The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information not corrosive to the skin. Criteria used for interpretation of results: EU
Conclusions:
The test item was not corrosive to skin.