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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 July to 30 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. This study is conducted according to an appropriate validated in vitro guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. In addition, the data is considered to be adequate and reliable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

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: EU Guideline Testing of Chemicals B46
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(2010)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimagnesium bis(orthophosphate)
EC Number:
231-824-0
EC Name:
Trimagnesium bis(orthophosphate)
Cas Number:
7757-87-1
Molecular formula:
Mg3O8P2
IUPAC Name:
trimagnesium bis(orthophosphate)
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult donors
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ model kit
- Tissue batch number(s): not specified
- Production date: not specified
- Shipping date: not specified
- Delivery date: 12 June 2012
- Date of initiation of testing: exposure: 11 July 2012; assessment of repsonse: 26 July

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco's Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT solution
- Incubation time: 3 hours at 37°C
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: without reference filter

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA
For the test item, the relative mean tissue viabilities obtained after the 15 min exposure period followed by the 42 h post-exposure incubation period were compared to the mean of the negative control tissues (n = 3). The relative mean viabilities were calculated as follows:

Relative mean viability (%) = (mean OD540 of test item / mean OD540 of negative control) x 100

Classification of irritation potential was based upon relative mean tissue viability following the 15 min exposure period followed by the 42 h post-exposure incubation period according to the following criteria:

Relative mean tissue viability ≤ 50%: Irritant (Category 2 according to Regulation (EC) No. 1272/2008 (CLP) and UN-GHS)
Relative mean tissue viability ≥ 50%: Non-irritant (Not classified according to Regulation (EC) No. 1272/2008 (CLP) and UN-GHS)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 mg

VEHICLE
-no vehicle used

NEGATIVE CONTROL
- Amount(s) applied: 10 µL Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+


POSITIVE CONTROL
- Amount(s) applied: 10 µl sodium dodecylsulfate
- Concentration: 5% (w/v)
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
triplicates

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 min
Value:
118.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
8.6% tissue viability
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viability values and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.The relative mean viability of the test item treated tissues was 118.8% after a 15-minute exposure period.

Any other information on results incl. tables

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 8.6% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.0%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.659 and the standard deviation value of the percentage viability was 6.2%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 7.6%. The test item acceptance criterion was therefore satisfied.

Table1 : Mean OD540 Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material

OD540of tissues

Mean OD540of triplicate tissues

±SDof OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Material¤

0.666

0.659

0.041

101.1

100*

6.2

0.696

105.6

0.615

93.3

Positive Control Material¤

0.059

0.056

0.006

9.0

8.6

1.0

0.061

9.3

0.049

7.4

Test Material

0.823

0.783

0.050

124.9

118.8

7.6

0.727

110.3

0.799

121.2


SD=    Standard deviation

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

¤ = Control group shared with Harlan Laboratories Ltd, Project numbers 41200853, 41200860, 41200866, 41200871, 41200880, 41200884 and 41201543


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was considered to be Non-Irritant (NI).
This study is conducted according to an appropriate validated in vitro guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. In addition, the data is considered to be adequate and reliable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). Trimagnesium bis(orthophosphate) is not considered to be classified in accordance with Regulation (EC) No. 1272/2008 (EU CLP).