Iron orthophosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 15 November 2011 and 18 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

13 April 2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

Study designed to be compatible with this guideline, however the guideline is not referred to in the study report.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Iron orthophosphate

EC Number:

233-149-7

EC Name:

Iron orthophosphate

Cas Number:

10045-86-0

Molecular formula:

FePO4

IUPAC Name:

iron(3+) phosphate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN(TM) Model Kit 0.38 cm² (SkinEthic Laboratories, Nice, France)
- Tissue batch number(s): not reported
- Production date: not reported
- Shipping date: not reported
- Delivery date: 15 November 2011
- Date of initiation of testing: 09 November 2011

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 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.
- Observable damage in the tissue due to washing: no

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

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive (H314, Category 1A) to skin if the viability after 3 minutes exposure is less than 35%.
- The test substance is considered to be corrosive (H314, Category 1B) to skin if the viability after 3 minutes exposure is greater or equal to 35% or if the viability after 60 minutes exposure is less than 35%.
- The test substance is considered to be corrosive (H314, Category 1C) to skin if the viability after 60 minutes exposure is greater or equal to 35%, or if the viability after 240 minutes exposure is less than 35%.
- The test substance is considered to be non-corrosive to skin if the viability after 240 minutes exposure is greater than or equal to 35%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg (100 μL of 0.9% w/v sodium chloride solution was added for wetting of the test item.)

NEGATIVE CONTROL
- Amount(s) applied: 50µL
- Concentration: 0.9% w/v

POSITIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

3, 60 & 240 minutes

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

110.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

96.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240 minute exposure

Value:

87.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
- Acceptance criteria met for positive control:
- Acceptance criteria met for variability between replicate measurements:
- Range of historical values if different from the ones specified in the test guideline:

Any other information on results incl. tables

RESULTS

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.

Test Item, Positive Control Item and Negative Control Item

Mean OD₅₄₀ values and viabilities for the negative control, positive control item and test item are given in Table 1.

The relative mean viability of the test item treated tissues was as follows:

240 minutes exposure                      :          87.1 %

60 minutes exposure                        :          96.4 %

3 minutes exposure                          :          110.8 %

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 3.1 % relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied.

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

Item	Exposure Period	Mean OD540of duplicate tissues	Relative mean viability (%)
Negative Control Item	240 Minutes	0.194	100*
Positive Control Item	240 Minutes	0.006	3.1
Test Item	240 Minutes	0.169	87.1
	60 Minutes	0.187	96.4
	3 Minutes	0.215	110.8

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

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Conclusions:

The test item was considered to be Non-Corrosive to the skin.

Executive summary:

Introduction: The purpose of this test is to evaluate the corrosivity potential of the test item using the EPISKIN^TM in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to be compatible with the following:

• OECD Guideline for the Testing of Chemicals No. 431 “In VitroSkin Corrosion: Human Skin Model Test” (adopted 13 April 2004)
• Method B.40 of CommissionRegulation (EC) No. 440/2008

The EPISKIN^TM model is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) chemicals.

Methods: 

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density (OD) was measured at 540 nm (OD₅₄₀).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results: 

The relative mean viability of the test item treated tissues was:

240 minutes exposure	:	87.1%
60 minutes exposure	:	96.4%
3 minutes exposure	:	110.8%

Quality criteria: 

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion:  The test item was considered to be Non-Corrosive to the skin.