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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-10-23 to 2012-10-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2004-04-13
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method 40 bis: In vitro skin corrosion: human skin model test
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2012-11-30

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimanganese bis(orthophosphate)
EC Number:
237-997-9
EC Name:
Trimanganese bis(orthophosphate)
Cas Number:
14154-09-7
Molecular formula:
Mn3O8P2
IUPAC Name:
trimanganese bis(orthophosphate)
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): trimanganese bis(orthophosphate)
- Physical state: pink cristalline solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: room temperature in the dark

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: human-derived epidermal keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Details on animal used as source of test system:
not applicable
Justification for test system used:
Validation studies have shown that tests employing human skin models are able to discriminate between known skin corrosives and non-corrosives.
Vehicle:
other: 0.9% w/v sodium chloride solution
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin TM tissues (purchased from SkinEthic Laboratories, Lyon, France)
- Batch No.: 13-EKIN-039
- Delivery date: 2012-10-23
- Date of initiation of testing: 2012-10-24 (following overnight pre-incubation)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: approx. 37 °C (incubated overnight)
- Temperature used during treatment / exposure: approx. 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the exposure period the tissues were removed from the the plate and rinsed with DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT solution (2.0 mL/well)
- Incubation time: 3 hours
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 uand the epidermis was separated from the collagen matrix. Both parts (epidermis and collagen matrix) were placed into micro tubes containing acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed to produce a homogenous coloured solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL 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 a microplate reader.

TEST FOR DIRECT MTT REDUCTION
The test item should be evaluated for their potential to interfere with MTT assay. 20 mg of the test item was added to 2.2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at room temperature for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue relative to the control, the test item was presumed to have reduced the MTT.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: tissues pass analysis for tissue functionality
- Contamination: absence of bacteria, fungus and mycoplasma as well as absence of HIV1 antibodies, HIV2 antibodies, Hepatitis B antigen, Hepatitis C antibodies
Please also refer to the field "Attached background material" below.

QUANTITATIVE MTT ASSESSMENT
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 remative mean viabilities were calculated in the following way: relative mean viability (%) = (mean OD540 of test item / mean OD540 of negative control) x 100

PREDICTION MODEL / DECISION CRITERIA
Classification of corrosivity potential was based on relative viabilities for each exposure time according to the prediction model as shown in the field "Any other information on materials and methods incl. tables" below (Table 1).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 20 mg of the solid test item

VEHICLE
- Amount(s) applied: 100 μl of 0.9% w/v sodium chloride solution

NEGATIVE CONTROL
- Amount(s) applied: 50 µL of 0.9% w/v sodium chloride solution

POSITIVE CONTROL
- Amount(s) applied: 50 µL of glacial acetic acid
Duration of treatment / exposure:
3, 60 and 240 minutes
Duration of post-treatment incubation (if applicable):
not applicable
Number of replicates:
Test item: duplicate
Negative control: duplicate
Positive control: duplicate

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean)
Run / experiment:
3 minute exposure
Value:
115.8
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
not examined
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean)
Run / experiment:
60 minute exposure
Value:
102.1
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
not examined
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean)
Run / experiment:
240 minute exposure
Value:
102.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS
- Direct MTT reduction: the MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.

ACCEPTANCE OF RESULTS
- Acceptance criteria met for positive control: the relative mean tissue viability for the positive control treated tissues was 2.5% relative to the negative control treated tissues following the 240-Minute exposure period (acceptibility criteria: relative mean tissue viability for the positive control should be 0 to 20 % relative to the negative control following 240 minute exposure).
- Acceptance criteria met for negative control: the mean OD540 for the negative control treated tissues was 0.812 (acceptability criteria: ≥ 0.600 and ≤ 1.500).

Any other information on results incl. tables

Table 3: Results after treatment with trimanganese bis(orthophosphate) and the controls

Dose Group

Exposure Interval

Mean OD540 of individual tissue 1

Mean OD540 of individual tissue 2

Mean Absorbance (OD540) of duplicate tissues

Rel. mean viability (%)

Test Item

3 minutes

0.955

0.925

0.940

115.8

Test Item

60 minutes

0.800

0.858

0.829

102.1

Negative Control

240 minutes

0.767

0.856

0.812

100*

Positive Control

0.015

0.025

0.020

2.5

Test Item

0.846

0.811

0.829

102.1

* The mean viability of the negative control tissues is set at 100%.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not corrosive to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not corrosive to the skin.