Inositol phosphates

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 08, 2009 to July 10, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The procedures performed in this study were based on OECD Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004), approved by the ECVAM Scientific Advisory Committee (ESAC). The GLP compliance is claimed through compliance with UK GLP standards [Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)].

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004)

Deviations:

yes

Remarks:

Interpretation of results

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Inositol phosphates

EC Number:

700-206-7

Molecular formula:

As this substance is a UVCB substance and consists varying degree of components, a single molecular formula is not applicable

IUPAC Name:

Inositol phosphates

Constituent 2

Reference substance name:

Synthetic inositol phosphates

IUPAC Name:

Synthetic inositol phosphates

Details on test material:

- Name of test material (as cited in study report): Synthetic inositol phosphates
- Physical state: Pale amber coloured liquid
- Lot/batch No.: 95816206JH-014
- Storage condition of test material: Room temperature in the dark

Test animals

Species:

other: Not applicable

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

Not applicable

Test system

Type of coverage:

other: Not applicable

Preparation of test site:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: Not applicable

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL

Duration of treatment / exposure:

3, 60 and 240 min.

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

PRE-TEST:

TEST FOR DIRECT MTT REDUCTION: 50 μL of the test material 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 h. Untreated MTT solution was used as a control.

PRE-INCUBATION (DAY 0: TISSUE ARRIVAL): 2.2 mL of maintenance medium, warmed to approximately 37 ºC, was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units/plate). A different 12-well plate was used for each test material, control and time point. The tissues were incubated at 37 ºC, 5 % CO2 in air for at least 24 h.

MAIN TEST:

APPLICATION OF TEST MATERIAL AND RINSING (DAY 1): 2.2 mL of assay medium, warmed to approximately 37 ºC, 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 material for exposure periods of 3, 60 and 240 min. Duplicate tissues were treated with the positive and negative control materials for an exposure period of 240 min. 50 μL of the test material was applied topically to the corresponding tissues ensuring uniform coverage of the tissues.

Negative control: Duplicate tissues, treated with 50 μL of 0.9 % w/v sodium chloride solution served as negative controls.
Positive control: Duplicate tissues, treated with 50 μL 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. 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 material. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (DAY 2): At the end of the formazan extraction period, each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored 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 the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: Relative tissue viability (% of negative control)

Value:

114

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 min. Reversibility: other: Not applicable. Remarks: None. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: Relative tissue viability (% of negative control)

Value:

45

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 min. Reversibility: other: Not applicable. Remarks: None. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: Relative tissue viability (% of negative control)

Value:

12.3

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 240 min. Reversibility: other: Not applicable. Remarks: None. (migrated information)

In vivo

Irritant / corrosive response data:

The relative mean viability of the test material treated tissues was as follows:
- 240 min. exposure: 12.3 %
- 60 min. exposure: 45.0 %
- 3 min. exposure: 114.0 %

Both treated tissues following the 3 min. exposure period and one treated tissue following the 60 min. exposure period appeared blue, which was considered to be indicative of viable tissue. One treated tissue following the 60 min. exposure period appeared blue/white which was considered to be indicative of semi-viable tissue. Following the 240 min. exposure period the treated tissues appeared white which was considered to be indicative of dead tissue.

Other effects:

Direct MTT reduction: The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.
Positive control: The relative mean tissue viability for the positive control tissues was 19.3 % relative to the negative control tissues following the 240 min. exposure period. The positive control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1: Mean OD540 values and viabilities for the negative control, positive control and test material

Material	Exposure period (min.)	Mean OD540 of duplicate tissues	Relative mean % viability
Negative control material	240	0.171	100*
Positive control material	240	0.033	19.3
Test material	240	0.021	12.3
	60	0.077	45
	3	0.195	114

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

⊕ = Control group shared with Harlan Laboratories Ltd Project number 2787/0008

Table 2: Qualitative evaluation of tissue viability (MTT uptake visual evaluation)

Material	Exposure period	Tissue 1	Tissue 2
Negative control material	240	-	-
Positive control material	240	++	++
Test material	240	++	++
	60	-	+
	3	-	-

MTT visual scoring scheme of SkinEthic tissues:

 - = Blue tissue (viable)

+ = Blue/white tissue (semi-viable)

++ = Tissue completely white (dead)

⊕ = Control group shared with Harlan Laboratories Ltd Project number 2787/0008

Applicant's summary and conclusion

Interpretation of results:

corrosive

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material satisfies the criteria for classification as corrosive to the skin according to EU labelling regulations Commission Directive 2001/59/EC.

Executive summary:

A study was conducted to evaluate the corrosivity potential of synthetic inositol phosphates using the EPISKIN^TMin vitro Reconstituted Human Epidermis (RHE) model after treatment periods of 3, 60 and 240 min. This method was designed to meet the requirements of the OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004).

 

Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 min. At the end of the exposure period, the test material 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 microtubes 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 was measured at 540 nm. Data are presented in the form of % viability (MTT reduction in the test material treated tissues relative to negative control tissues).

 

The relative mean viability of the treated tissues was 114.0, 45.0 and 12.3% after 3, 60 and 240 min. of exposure, respectively.

 

The test material satisfies the criteria for classification as corrosive to the skin according to EU labelling regulations Commission Directive 2001/59/EC. The symbol “C”, the indication of danger “Corrosive” and the risk phrase R 34 “Causes burns” are therefore required. The UN packing group III is also required.