Dizinc pyrophosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Jun-14 Jul 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health and Consumer Protection, Hamburg, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Dizinc pyrophosphate

EC Number:

231-203-4

EC Name:

Dizinc pyrophosphate

Cas Number:

7446-26-6

Molecular formula:

H4O7P2.2Zn

IUPAC Name:

dizinc(2+) (phosphonooxy)phosphonate

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation being used as a replacement for the Draize Skin irritation test. Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The EpiDerm™ model
- Tissue batch number(s): EPI-200, Lot no. 25829
- Date of initiation of testing: 20 June 2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: All tissues were washed carefully with DPBS after the exposure period.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues/killed tissues: killed tissues
- Procedure used to prepare the killed tissues: incubating in distilled water at 37 °C for 42 h
- N. of replicates: 3
- Method of calculation used: direct comparison of treated to untreated water-killed tissues

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be a non-irritant to skin if the viability of three individual tissues after 60 minutes exposure period to the test substance followed by 42 hours post-exposure incubation is greater than 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- OTHER EFFECTS:
- Direct-MTT reduction: The results of the direct interference evaluation procedure showed that no interference due to direct reduction of MTT by the test substance occurred.
- Colour interference with MTT: An assessment found the test item had no potential to cause colour interference with the MTT endpoint. It was therefore considered unnecessary to use the results of the colour correction tissues for quantitative correction of results or for reporting purposes

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg applied uniformly over an area of 0.63 cm²

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): DPBS 30 µL
- Concentration (if solution): 100%

POSITIVE CONTROL
- Amount(s) applied (volume or weight): aqueous sodium dodecyl sulphate 30 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

35 min at 37 °C and 25 min at RT

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-min exposure followed by 42-hour post-exposure incubation period

Value:

101.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

6.5%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD540 for the negative control treated tissues was 1.396 and the standard deviation determined for all triplicates was below 18%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 6.5% relative to the negative control treated tissues. The positive control acceptance criteria was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The relative mean tissue viability for the test item treated tissues was 101.6% of the negative controls after a 60-min exposure period and 42-hour post-exposure incubation period. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was <18%. The test item acceptance criteria was therefore satisfied.

Any other information on results incl. tables

 Mean OD540 Values and Viabilities for the Negative Control, Positive Control, and Test Items

Item	OD540 of tissues	Mean OD540 of triplicate tissues	%CV of mean OD540	Relative individual tissue viability (%)	Relative mean viability (%)	%CV of Relative mean viability (%)
Negative Control	1.313	1.396	7.6	94.1	100*	7.6
	1.360			97.4
	1.516			108.6
Positive Control	0.083	0.092	10.4	5.9	6.5	10.9
	0.090			6.4
	0.102			7.3
Test Item	1.349	1.418	5.3	96.6	101.6	5.2
	1.408			100.9
	1.497			107.2

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

OD = Optical density

CV = Coefficient ovf variation

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008