Bisguanidinium phosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018/11/07 - 2018/11/09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Bisguanidinium phosphate

EC Number:

226-552-4

EC Name:

Bisguanidinium phosphate

Cas Number:

5423-23-4

Molecular formula:

CH5N3.1/2H3O4P

IUPAC Name:

Diguanidinium hydrogen phosphate

Test material form:

solid: particulate/powder

In vitro test system

Test system:

isolated skin discs

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

other: not applicable (human)

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Test system

Type of coverage:

other: not applicable (in vitro test system)

Preparation of test site:

other: The epidermal surface was first moistened with 5 μL deionised water

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5 %

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

Duration of treatment / exposure:

15 min

Observation period:

42 h

Number of animals:

not applicable (in vitro test system)

Details on study design:

Exposure (day 0)
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.

Rinsing (day 0)
After the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

Post-incubation (day 0-2)
After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37±1°C in an incubator with 5±1 % CO2, ≥95% humidified atmosphere.

MTT test after 42 hours incubation (day 2)
After the 42 hours (± 1h) incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, ≥95% humidified atmosphere.

Formazan extraction (day 2)
At the end of incubation with MTT a formazan extraction was undertaken:
A disk of epidermis was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 μL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for four hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

Cell viability measurements (day 2)
Following the formazan extraction, 2×200 μL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at 570 nm (±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using acidified isopropanol solution as the blank (6×200 μL).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

103

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

110

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

105

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

106

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is white and therefore considered to be not able to significantly stain the tissues and lead false estimate of viability. Furthermore, the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean OD value of the three negative control tissues was 0.880.
- Acceptance criteria met for positive control: Yes, the mean OD value obtained for the positive control was 0.088 and this result corresponds to 10 % viability when compared to the results obtained from the negative control.
- Acceptance criteria met for variability between replicate measurements: Yes, each calculated standard deviation value (SD) for the % viability was below 18.

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

In this in vitro skin irritation test using the EPISKIN model, the test item bisguanidinium phosphate did not show significantly reduced cell viability in comparison to the negative control (mean value: 106 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item is considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. Thefore the experiment is evaluated to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item bisguanidinium phosphate is considered to be non-irritant to skin and has not to be classified for skin irritation (UN GHS No Category).

Executive summary:

EpiSkin^TM SM test of bisguanidinium phosphate was performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1°C for 42 hours (± 1h) in an incubator with 5±1% CO₂, ≥ 95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1°C in 5±1% CO₂, ≥ 95% humidified atmosphere, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item bisguanidinium phosphate did not show significantly reduced cell viability in comparison to the negative control (mean value: 106 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item is considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. Thefore the experiment is evaluated to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item bisguanidinium phosphate is considered to be non-irritant to skin and has not to be classified for skin irritation (UN GHS No Category).