Polyphosphoric acids, ammonium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 January 2015 to 19 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conclusive, performed to a valid guideline (OECD TG 439, adopted 22 July 2010) and was conducted under GLP conditions. No deviations from the test methods were noted.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP Inspection: 17 June 2015 Date of Signature on Certificate: 24 September 2015

Test material

Test material information

Constituent 1

Reference substance name:

Polyphosphoric acids, ammonium salts

EC Number:

269-789-9

EC Name:

Polyphosphoric acids, ammonium salts

Cas Number:

68333-79-9

Molecular formula:

[NH4PO3]n

IUPAC Name:

undecaammonium bis(phosphonatooxy)phosphinate dihydrogen phosphate hydrogen (phosphonatooxy)phosphonate hydrogen phosphate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): IP 65: POLYPHOSPHORIC ACIDS, AMMONIUM SALTS (SOLID)
- Physical state: white powder
- Analytical purity: 100%
- Lot/batch No.: MD141010
- Expiration date of the lot/batch: 14 October 2015
- Storage condition of test material: Store over silica gel at room temperature in the dark

Test animals

Species:

other: reconstructed human epidermis

Strain:

other: reconstructed human epidermis

Details on test animals or test system and environmental conditions:

EpiSkin™ Reconstructed Human Epidermis Model Kit

The EPISKIN™ model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Supplier : SkinEthic Laboratories, Lyon, France
Date received : 13 January 2015
EpiSkinTM Tissues (0.38cm2) lot number : 15-EKIN-002
Maintenance Medium lot number: 15-MAIN3-002
Assay Medium lot number : 15-ESSC-002

Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory : Yes
Temperature Indicator Color Satisfactory : Yes
Agar Medium Color Satisfactory : Yes

Adaptation to cell culture conditions (pre-incubation): 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first
column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item.

Incubation Conditions:
37 °C, 5% CO2 in air overnight.

Test system

Type of coverage:

other: open in vitro system

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

water

Controls:

other: Positive and negative control items were used

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 10 mg (26.3 mg/cm2)

VEHICLE
- Amount(s) applied (volume or weight with unit): 5 µL of sterile distilled ater was topically applied to the epidermal surface prior to applying the tst item in order to improve contact between the test item and the epidermis

NEGATIVE CONTROL ITEM:

Identification: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
Batch: 1553513
Purity: Not supplied
Expiry Date: 01 March 2017
Storage Conditions: Approximately 4°C in the dark

POSITIVE CONTROL ITEM:
Identification: Sodium Dodecyl Sulphate (SDS)
Batch: 1294323
Purity: Not supplied
Expiry Date: 25 October 2017
Storage Conditions: Room temperature

The negative control item, DPBS, was used as supplied.

The positive control item, SDS, was prepared as a 5% w/v aqueous solution.

Duration of treatment / exposure:

15 minutes

Observation period:

Not applicable. The tissues were transferred to the second column of 3 wells containign 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

Number of animals:

Not applicable. The test was performed in triplicates for each treatment and control group.

Details on study design:

PREPARATION OF MTT AND ACIDIFIED ISOPROPANOL

A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.

A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.

REMOVAL OF TEST SUBSTANCE
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 DPBS to gently remove any residual test item.

ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT

The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.

If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

MTT LOADING/FORMAZAN EXTRACTION (DAY 3 OF MAIN TEST)

Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.

2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. 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 made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of
acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (DAY 6):
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-labeled 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 562 nm (without a reference filter) using the Anthos 2001 microplate reader

INTERRETATION OF RESULTS

Quantitative MTT Assessment (Percentage Tissue Viability):
For the test item the relative mean tissue viabilities obtained after the 15-Minute exposure period 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:

Relative mean viability (%) = (mean OD562 of test item/mean OD562 of negative control) x 100

ASSAY ACCEPTANCE CRITERIA:

The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:

Positive Control
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤18.

Negative Control
The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues was ≥0.6, and the standard deviation value of the percentage viability is ≤18.

Test Item
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: relative mean tissue viability (%)

Value:

114.1

Remarks on result:

other:

Remarks:

Basis: other: mean value test item. Time point: 15 minute exposure. Max. score: 100.0. Reversibility: other: not applicable. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: relative mean tissue viability (%)

Value:

28.6

Remarks on result:

other:

Remarks:

Basis: other: mean value positive control. Time point: 15 minute exposure. Max. score: 100.0. Reversibility: other: not applicable. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: relative mean tissue viability

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: mean value positive control item. Time point: 15 minute exposure. Max. score: 100.0. Reversibility: other: not applicable. Remarks: The mean viability of the negative control tissues is set at 100%. (migrated information)

In vivo

Irritant / corrosive response data:

The individual and mean OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1 below. The mean viabilities 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 114.1% after a 15-Minute exposure period and 42 hours post-exposure incubation period.

It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

Any other information on results incl. tables

Table 1: Mean OD562Values and Percentage Viabilities for the Negative Control Item, Positive

Control Item and Test Item

Item	OD562of tissues	Mean OD562of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean tissue viability (%)	± SD of Relative mean viability (%)
Negative control item	0.677	0.675	0.02	100.3	100*	3.5
	0.697			103.3
	0.651			96.4
Positive control item	0.207	0.193	0.02	30.7	28.6	3.5
	0.166			24.6
	0.206			30.5
Test item	0.770	0.770	0.05	114.1	114.1	7.7
	0.718			106.4
	0.822			121.8

 

SD = Standard deviation

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

OD = Optical density

Direct MTT Reduction

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 28.6% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 3.5%. The positive control acceptance criterion was therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.675 and the standard deviation value of the percentage viability was 3.5%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues was 7.7%. The test item acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was classified as non-irritant.

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2- yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. 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-labeled 96-well plate. The optical density was measured at 562 nm. 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 114.1% after the 15-Minute exposure period and 42 hours post-exposure incubation period.

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

Conclusion

The test item was considered to be non-irritant.

Therefore, classification according to EU DSD (Council Directive 67/548/EEC), EU CLP (Regulation (EC) No 1272/2008) and UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) is not required.