Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 06 November 2012 and 12 November 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspectection: 10 July 2012, Date of Signature: 30 November 2012

Test material

Constituent 1
Chemical structure
Reference substance name:
Magnesium bis(dihydrogenorthophosphate)
EC Number:
236-004-6
EC Name:
Magnesium bis(dihydrogenorthophosphate)
Cas Number:
13092-66-5
Molecular formula:
H3O4P.1/2Mg
IUPAC Name:
magnesium hydrogen phosphate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Sponsor's identification : IP 31: Magnesium bis(dihydrogenorthophosphate)
EC number : 236-004-6
CAS number : 13092-66-5
Description : white powder
Batch number : Laboratory preparation 11.10.11
Purity : >98%
Date received : 02 October 2012
Expiry date : not supplied
Storage conditions : room temperature in the dark

Test animals

Species:
other: reconstituted human epidermis model
Strain:
other: reconstituted human epidermis model
Details on test animals or test system and environmental conditions:
Not applicable

Test system

Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
Controls:
no
Amount / concentration applied:
TEST MATERIAL

- The test material was applied neat.

- Amount(s) applied (volume or weight with unit):
Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 μl of sterile distilled water to improve contact between the solid test item and the epidermis.

- Concentration (if solution):
The test material was used as supplied.

VEHICLE
No vehicle used
Duration of treatment / exposure:
15 minute exposure & 42 hour post-exposure incubation
Observation period:
Not applicable
Number of animals:
Triplicate tissues were treated
Details on study design:
TEST SITE
- Area of exposure:
Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 μl of sterile distilled water to improve contact between the solid test item and the epidermis.

- % coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

- Type of wrap if used:
None used

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS 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 material. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37ºC, 5% CO2 in air for 42 hours.

- Time after start of exposure:
15 minutes post-exposure

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
For the test material the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

mean OD540 of test material / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is ≤50% : Irritant: H315, Category 2

Mean tissue viability is >50% : Non-Irritant (NI)

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
86.2
Other effects / acceptance of results:
The relative mean viability of the test material treated tissues was 86.2% after a 15-minute exposure.

Any other information on results incl. tables

RESULTS

Direct MTT Reduction

The MTT solution containing the test material did not turn blue which indicated that the test material did not directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD540 values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test material treated tissues was 82.6% after a 15 minute exposure.

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 5.4% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 2.2%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.697 and the standard deviation value of the percentage viability was 8.2%. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 3.4%. 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¤

0.728

0.697

0.057

104.4

100*

8.2

0.631

90.5

0.732

105.0

Positive Control Material¤

0.032

0.038

0.015

4.6

5.4

2.2

0.055

7.9

0.026

3.7

Test Material

0.588

0.601

0.024

84.4

86.2

3.4

0.628

90.1

0.586

84.1


SD=    Standard deviation

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

¤ = Control group shared with Harlan Laboratories Ltd, Project numbers 41205113, 41205115, 41205120, 41205125, 41205128 and 41205133


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). Magnesium bis(dihydrogenorthophosphate) is not considered to be classified in accordance with Regulation (EC) No. 1272/2008 (EU CLP).