Amidinourea 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:

08-05-2017 to 03-07-2017

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:

OECD Guideline for the Testing of Chemicals No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Amidinourea phosphate

EC Number:

241-659-6

EC Name:

Amidinourea phosphate

Cas Number:

17675-60-4

Molecular formula:

C2H6N4O.xH3O4P

IUPAC Name:

diaminomethylideneurea;phosphoric acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Batch No.: 16VL8189
Expiry Date: 03 August 2017
Storage Conditions: room temperature, protected from light

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm™ reconstructed human epidermis model (MatTek)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM, a reconstituted three-dimensional human epidermis model.

Source strain:

not specified

Details on animal used as source of test system:

EpiDerm™ reconstructed human epidermis model (MatTek)

Justification for test system used:

This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS “Category 2”. Depending on the regulatory framework it can also be used to identify non-classified chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

The test was carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell ). The EpiDerm epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.

Preparation and Application of the Test Item

25 μL of sterile DPBS was applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm2) of the test item was applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue by gently shaking the inserts or by using a bulbheaded Pasteur pipette.

Controls
Controls were set up in parallel to the test item in order to confirm the validity of the test.

Negative Control
Dulbecco’s phosphate buffered saline (DPBS; Gibco, Cat. No. 14040-091, Lot No.: 1838067).

Positive Control
5% sodium dodecyl sulfate in H2O (TC-SDS-5%; Applichem CAS No.: 151-21-3, Lot No.: 40015277)

Dose Groups
1. Negative control 30 μL DPBS
2. Positive control 30 μL 5% SDS solution
3. Test Item 25 mg + 25 μL DPBS
The test was performed on a total of 3 tissues per dose group.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg (39 mg/cm2)

Duration of treatment / exposure:

Pre-incubation: 18 ± 3 h.
After dosing: 35 ± 1 min.
Post-incubation: 24 ± 2 h. Following this the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for an additional 18 ± 2 h.

Duration of post-treatment incubation (if applicable):

See above

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: cytotoxic effects via MTT reduction assay.

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Within experiment quality criteria met.

Any other information on results incl. tables

Results of the Test Item GUP

Name NK PC TM Tissue 1 2 3 1 2 3 1 2 3 absolute OD550 2.221 2.078 2.139 2.065 1.804 1.801 0.130 0.132 0.140 0.139 0.128 0.134 1.956 1.907 1.856 1.818 1.963 1.925 OD550(blank- corrected) 2.179 2.036 2.097 2.023 1.762 1.759 0.089 0.090 0.098 0.097 0.086 0.092 1.914 1.865 1.814 1.776 1.921 1.883 mean OD550of the duplicates (blank- corrected) 2.107 2.060 1.761 0.089 0.098 0.089 1.890 1.795 1.902 total mean OD550of 3 replicate tissues (blank-corrected) 1.976* 0.092 1.862 SD OD550 0.188 0.005 0.059 relative tissue viability [%] 106.6 104.3 89.1 4.5 5.0 4.5 95.6 90.8 96.2 mean relative tissue viability [%] 100.0 4.7** 94.2 SD tissue viability [%]*** 9.5 0.2 3.0 CV [% viabilities] 9.5 5.3 3.1 * Blank-corrected mean OD570nm of the negative control corresponds to 100% absolute tissue viability. ** Mean relative tissue viability of the three positive control tissues is < 20%. *** Standard deviation (SD) obtained from the three concurrently tested tissues is < 18% NK negative control PC positive control

Quality Criteria       

	Value	Cut off	pass/fail
Mean Absolute OD570nmNK	1.976	0.8 ± NK ± 2.8	pass
Relative Viability [%] PC	4.7	± 20%	pass
SD Viability [%]	0.2 -9.5	± 18%	pass

NK negative control

PC positive control

Historical Data 

	^Mean OD570±30nm NK	Mean Relative Viability [%] PC	SD Viability [%]
Mean	1.831	3.9	4.4
SD	0.357	1.9	5.1
n	27	27	88

Historical data were generated from 2009 to 2016.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no irritant effects.

Executive summary:

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human epidermis model EpiDerm (MatTek) comprising a reconstructed epidermis with a functional stratum corneum. In the present study GUP was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (94.2%) after 60 min treatment and 42 h post-incubation. The controls confirmed the validity of the study.