Amidinourea phosphate

Endpoint summary

Description of key information

In vitro skin sensitisation

An in vitro KeratinoSens™ assay was used to detect potential skin sensitisation of the test item. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 μM. Under the condition of this study the result for the test item is negative.

An in vitro h-CLAT assay was used to detect potential skin sensitisation of the test item. The following concentration range was tested in the assay: 1000, 833, 694, 578, 482, 401, 334, 279 μg/mL.

Under the condition of this study the result for the test item is positive.

During the solubility test for the DPRA, it was detected that the test item was not soluble in any of the tested vehicles. Therefore, the test was not performed.

Due to the equivocal test result, it was decided to perform an in-vivo LLNA test addtionally.

In vivo skin sensitisation

An in vivo LLNA test was used to detect potential skin sensitisation of the test item. The following concentration range was tested: 10, 5, 2% (w/v). Under the condition of this study the result for the test item is negative.

Overall skin sensitisation conclusion

As the in-vitro tests showed equivocal results, the in-vivo LLNA test has been performed. Based on the in-vivo LLNA test, no skin sensitisation was shown.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20-03-2017 to 25-10-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

OECD Guidelines for Testing of Chemicals, number 442d “In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method” (adopted: February 04, 2015)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers was reported by several studies and represents the second key event of the skin sensitisation process as described by the adverse outcome pathway. Therefore the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Specific details on test material used for the study:

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

Details on the study design:

The test was carried out using the transgenic cell line KeratinoSens™ a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 8 experiment 1; P 6 experiment 2) were used. Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 1°C and 5% CO2. For test material exposure, cells were cultured in medium.

Experimental Procedure
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability. After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 μL test item exposure medium. 50 μL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item. All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS (Gibco Life Science; Lot No.: 1839805 (exp. 1)/1813255 (exp. 2)). Subsequently 20 μL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light. Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 μL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 μL test item exposure medium. 27 μL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 μL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1) or over the weekend (experiment 2). After the incubation period the plate was shaken for 10 min and the OD was measured at = 600 nm.

Positive control results:

Valid: The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.44 in experiment 1; 4.28 in experiment 2). The calculated EC15 was between 7 and 34 µM (21.80 µM in experiment 1; 32.87 µM in experiment 2).

Key result

Run / experiment:

other: First & second experiments : EC 5 values µM

Parameter:

other: Activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™.

Value:

1 000

Vehicle controls validity:

other: Solvent controls: valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Positive response at 64 µM

Remarks on result:

no indication of skin sensitisation

Remarks:

the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs.

A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.

Blank

A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.

Negative Control

DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.

Positive Control

Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%;) was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 μM – 64 μM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

Interpretation of results:

study cannot be used for classification

Conclusions:

Data generated with the present Test Guideline should be considered in the context of integrated approaches, such as IATA, combining them with other complementary information e.g. derived from in vitro assays

Executive summary:

An in vitro KeratinoSens™ assay was used to detect potential skin sensitisation of the test item. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 μM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. In the first experiment, at a test item concentration of 2000 μM, the cell viability was 116.2%. Only at this concentration was there a significant luciferase induction: >1.5 was found. The calculated EC1.5 was > 1000 μM (1048.62 μM).

In the second experiment, at the highest concentration the cell viability was 148.4%. Only at this concentration was there significant luciferase induction: >1.5 was found. The calculated EC1.5 was > 1000 μM (1739.82 μM).

A slight dose response for luciferase activity was observed for each individual run as well as for an overall increase in luciferase activity induction. Under the condition of this study the test item is considered as non sensitiser.

The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 μM was between 2 and 8 (2.44 in experiment 1; 4.28 in experiment 2). The calculated EC1.5 was between 7 and 34 μM (21.80 μM in experiment 1; 32.87 μM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (11.7% experiment 1; 19.7% experiment 2).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

06. August 2019 - 03. February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidline 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation

Version / remarks:

27 Jun 2018

Deviations:

yes

Remarks:

All deviations were considered not to have compromised the validity or integrity of the study.deviations were considered not to have compromised the validity or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic Dendritic Cell (DC) activation, which plays a critical role in T-cell priming. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The Relative Fluorescence Intensity of surface markers compared to solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitizers and non-sensitizers.

Specific details on test material used for the study:

Batch No.: 18VL8610
Expiry Date: 06 October 2020
Storage Conditions: room temperature

Details on the study design:

The THP-1 is an immortalized human monocytic leukemia cell line derived from an acute monocytic leukemia patient. The THP-1 cell line is obtained from ATCC (Ref: TIB-202, American Type Culture Collection, Manassas, USA) by the intermediate of LGC Standards (Molsheim, France).
The THP-1 cells are stored in a cryoprotective medium in a liquid nitrogen container.
Cells were grown using general culture procedures. They were cultured in cRPMI medium and maintained in a humidified incubator set at 37°C, 5% CO2 and were not allowed to exceed a cell density of 1 x 106 cells/mL or more than 30 passages.
The culture medium (cRPMI) was composed of RPMI 1640 with 10% FBS, 0.05 mM 2 mercaptoethanol and with penicillin and streptomycin.
During cell culturing, cell viability was checked using trypan blue.

Positive control results:

Valid: NiSO4 (final concentration in wells of 120 μg/mL) and DNCB (final concentration in wells of 4 μg/mL) the RFI values of both CD86 and CD54 met positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability was than 50%.

Key result

Run / experiment:

other: Full test up to 1000 µg/mL

Parameter:

other: RFI for CD86

Value:

150

Vehicle controls validity:

valid

Remarks:

NaCl

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

DNCB and NiSO4

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Full test up to 1000 µg/mL

Parameter:

other: RFI for CD54

Value:

200

Vehicle controls validity:

valid

Remarks:

NaCl

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

DNCB and NiSO4

Remarks on result:

positive indication of skin sensitisation

Vehicle and two positive controls were set up in parallel in order to confirm the validity of the test.

Vehicle controls

The vehicle controls were 0.2 % DMSO (as positive control DNCB was diluted with DMSO), 0,9 % NaCl (as positive control NiSO4 and test item were diluted with NaCl) and cell medium cRPMI. All controls were added to seeded cells and were included in determination of the background.

Positive controls

The positive control DNCB was prepared at the concentration of 8 μg/mL in DMSO before treatment (final concentration in wells of 4 μg/mL) as follows:

. on the treatment day, the required quantity of DNCB was mixed with DMSO at the concentration of 2 mg/mL,

. this solution was then 250-fold diluted in cRPMI in order to obtain a 8 μg/mL DNCB stock solution.

The positive control NiSO4 was prepared at the concentration of 240 μg/mL in 0.9% NaCl before treatment (final concentration in wells of 120 μg/mL) as follows:

. on the treatment day, the required quantity of NiSO4 was mixed with 0.9% NaCl at the concentration of 12 mg/mL,

. this solution was then 50-fold diluted in cRPMI in order to obtain a 240 μg/mL NiSO4 stock solution.

Interpretation of results:

study cannot be used for classification

Conclusions:

Data generated with the present Test Guideline should be considered in the context of integrated approaches, such as IATA, combining them with other complementary information e.g. derived from in vitro assays

Executive summary:

The objective of the study was to determine the ability of the test item to induce an increase in expression of cell surface markers in THP-1 cells using the h-CLAT test method.

The following concentration range was tested in the assay:

1000, 833, 694, 578, 482, 401, 334, 279 μg/mL.

Test item stock solutions were prepared at 8 different concentrations by 1.2-fold dilutions using the selected vehicle. The highest concentration corresponded to the highest achievable non-cytotoxic concentration as no CV75 was obtained.

All stock formulations were then 50-fold diluted into cRPMI to obtain working solutions. In parallel, the working solutions of both positive controls (DNCB and NiSO4) were prepared.

All working solutions were finally used for exposure by adding 500 μL of working solutions to the volume of THP-1 cell suspension in the plate (500 μL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate.

The treated plates were then incubated for 24 hours ± 30 minutes in a humidified incubator set at 37°C and 5% CO2.

At the end of the treatment phase, an inspection under a light microscope was performed to each well. Then, cells were transferred into sample tubes and collected by centrifugation, washed twice with 1 mL FACS buffer and blocked with 600 μL of blocking solution (see § Study plan adherence) and incubated at 4°C for 15 minutes (± 1 minute). After blocking, cells were split in three aliquots of 180 μL into a 96-well round bottom plate and centrifuged before staining with antibodies. A volume of 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies prepared in FACS buffer was added to each aliquot before incubation for 30 minutes (± 2 minutes) at 4°C.

Finally, cells were washed with 150 μL FACS buffer twice and re-suspended in 200 μL FACS buffer. The plate was then positioned into the plate-reader of the flow cytometer. A volume of 10 μL of PI solution at 12.5 μg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 μg/mL per well.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06. Dec 2019 - 03. Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

Deviations:

yes

Remarks:

The observed differences in the environmental parameters were considered not to adversely affect the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Batch No.: 18VL8610
Expiry Date: 06 October 2020
Storage Conditions: room temperature, protected from light

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Age of animals at starting: 9 weeks old
Body weight range at starting: 19.1 – 20.8 grams
Acclimatization time: 13 days
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
The minimum /maximum values of temperature and relative humidity were recorded twice every day during the acclimatization and experimental phases.

Vehicle:

dimethyl sulphoxide

Concentration:

25% (w/v)

No. of animals per dose:

4

Details on study design:

For the main assay, concentrations of 10% (w/v), 5% (w/v) and 2% (w/v) in DMSO were considered to be acceptable based on preliminary study results.
All mice were observed daily (until day 6) for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Results were interpreted according to OECD Guideline 429

Positive control results:

Valid: positive control substance produces a significant lymphoproliferative response increases (SI>3)

Key result

Parameter:

SI

Value:

2.8

Test group / Remarks:

10% (w/v) in DMS

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

5% (w/v) in DMS

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

2% (w/v) in DMS

Cellular proliferation data / Observations:

No mortality or systemic toxicity was observed during the main study. No test item residue was observed on the ears of the animals. There were no indications of any irritancy at the site of application.
No evidence of test item related effects were observed on the mean body weight changes in the main study

DPM. DPN and Stimulation Index Values for all Groups

Test Group Name	Animal Number	Measured DPM	Total DPM	No. of Nodes	DPN	Mean DPN	Stimulation Index Values
Background	-	36	-	-	-	-	-
(5 (w/v) % TCA)
Negative control (DMSO)	9372	482	446.0	2	223.0	391.0	1.0
	9378	558	522.0	2	261.0
	9373	1541	1505.0	2	752.5
	9381	691	655.0	2	327.5
GUP - Guanylurea Phosphate  10% (w/v) in DMSO	9377	1995	1959.0	2	979.5	1109.0	2.8
	9386	2807	2771.0	2	1385.5
	9383	1597	1561.0	2	780.5
	9376	2617	2581.0	2	1290.5
GUP - Guanylurea Phosphate  5% (w/v) in DMSO	9390	1117	1081.0	2	540.5	481.4	1.2
	9385	1133	1097.0	2	548.5
	9384	1022	986.0	2	493.0
	9389	723	687.0	2	343.5
GUP - Guanylurea Phosphate  2% (w/v) in DMSO	9374	965	929.0	2	464.5	510.0	1.3
	9388	1372	1336.0	2	668.0
	9379	1509	1473.0	2	736.5
	9387	378	342.0	2	171.0
Positive control (25% (w/v) HCA) in DMSO	9380	5377	5341.0	2	2670.5	3201.3	8.2
	9382	6174	6138.0	2	3069.0
	9391	9959	9923.0	2	4961.5
	9375	4244	4208.0	2	2104.0

Notes:

1. Total DPM = Measured DPM – Background DPM value of TCA

2. DPN = (Measured DPM – Background DPM value of TCA) / Number of nodes

Interpretation of results:

GHS criteria not met

Conclusions:

GUP - Guanylurea Phosphate, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidline 442C: In Chemico Skin sensitsation

Deviations:

yes

Remarks:

All deviations were considered not to have compromised the validity or integrity of the study.deviations were considered not to have compromised the validity or integrity of the study.

GLP compliance:

not specified

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Batch No.: 18VL8610
Expiry Date: 06 October 2020
Storage Conditions: room temperature

Details on the study design:

The aim of the study was to perform solubility assay with the test item in order to evaluate the applicability of the test item to the in vitro sensitization test methods: DPRA

Positive control results:

n/a

Remarks on result:

not determinable because of methodological limitations

Remarks:

The test item found not soluble, in none of the tested vehicle, despite a long period of sonication. Therefore the OECD 442C test was not performed.

Other effects / acceptance of results:

The test item found not soluble, in none of the tested vehicle, despite a long period of sonication. Therefore the OECD 442C test was not performed.

The aim of the study was to perform solubility assay with the test item in order to evaluate the applicability of the test item to the in vitro sensitization test methods: DPRA

The test item found not soluble, in none of the tested vehicle, despite a long period of sonication. No DPRA test has been performed

Interpretation of results:

other: test item found not soluble, no DPRA test has been performed

Conclusions:

The test item found not soluble, in none of the tested vehicle, despite a long period of sonication. No DPRA test has been performed

Executive summary:

The test item found not soluble, in none of the tested vehicle, despite a long period of sonication. No DPRA test has been performed

Reference 5

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because the available in vitro test methods are not applicable for the substance and therefore an in vivo skin sensitisation study was conducted

Justification for type of information:

Three different in vitro skin sensitisation studies (OECD 442 C/D and E) have been performed, but result was equivocal.
OECD 442 C: Test item was insoluble in all tested vehicles, no DPRA test has been performed
OECD 442 D: Result in KeratoSens was negative
OECD 442 E: Result in h-CLAT test was positive
As the overall result of the performed in-vitro tests was equivocal, it was decided to run the in-vivo LLNA test according to OECD 429.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

In vivo skin irritation

Under the conditions of this study the test item is not considered to be a skin sensitiser. No classification is proposed (Annex I of Regulation (EC) 1272/2008).