Amidinourea phosphate

Endpoint summary

Description of key information

Skin irritation_EpiDerm

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. 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.

Eye irritation_BCOP:

The eye irritancy potential of GUP was investigated in the bovine corneal opacity and permeability assay. The test item was suspended with physiological saline 0.9% NaCl (see 10.2) to give a 20% concentration. All 3 corneas treated with GUP showed slight opacity of the tissue. The following mean in vitro irritation score was calculated: 3.58. No prediction can be made regarding the classification of the test substance GUP according to the evaluation criteria.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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)

Specific details on test material used for the study:

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

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

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-03-2017 to 16-05-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

OECD Guideline for the Testing of Chemicals, number 437 “Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage” (adopted: 26 July 2013)

Deviations:

yes

Remarks:

minor deviations that did not influence the quality or integrity of the present study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

Species:

other: Bovine eyes were collected from a slaughterhouse.The eyes were examined for defects, defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera.

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 1 °C.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL of the test item applied (The test item was suspended with physiological saline 0.9% NaCl to give a 20% concentration.

Duration of treatment / exposure:

4 hours ± 5 minutes

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3 per group

Details on study design:

See any other information on materials and methods

Irritation parameter:

in vitro irritation score

Value:

3.58

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made regarding the classification of the test substance GUP according to the evaluation criteria.

In vitro irritation score

Cornea	Test Item	Corrected	Corrected OD490 Value	IVIS
No.		Opacity
1	Negative control	0.43	0.004
2		2.21	0.014
3		1.32	0.016
MV		1.32	0.011	1.49
4	Positive control	133.12	0.861
5		122.92	2.069
6		129.61	1.078
MV		128.55	1.336	148.59
7	Test Item	1.96	0.008
8		3.17	0.011
9		4.45	0.058
MV		3.2	0.025	3.58

MV = mean value

IVIS = in vitro irritation score

Interpretation of results:

GHS criteria not met

Conclusions:

No prediction can be made regarding the classification of the test substance GUP according to the evaluation criteria.

Executive summary:

The eye irritancy potential of GUP was investigated in the bovine corneal opacity and permeability assay. The test item was suspended with physiological saline 0.9% NaCl (see 10.2) to give a

20% concentration. All 3 corneas treated with GUP showed slight opacity of the tissue. The following mean in vitro irritation score was calculated: 3.58.

No prediction can be made regarding the classification of the test substance GUP according to the evaluation criteria.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation_EpiDerm

No irritation was evident. According to Annex I of Regulation (EC) 1272/2008 no classification is proposed for GUP.

Eye irritation_BCOP:

No prediction can be made regarding the classification of the test substance GUP according to the evaluation criteria.