Copper(2+), bis[N-[amino(imino-.kappa.N)methyl]urea-.kappa.O]-, nitrate (1:2)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03/15/16 to 08/09/2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study realised according to OECD GD 489 (2014) and GLP but the determination of Copper Guanylurea nitrate (CuGUN) in treatment formulations was performed by a non-GLP-compliant laboratory (ESCOM, France) using a method that was similar in its sensitivity for the dose level of copper to be assayed, to the one validated and used by the GLP-compliant laboratory INERIS. Analysis were performed with the presence of GLP-compliant INERIS study personnel and under the supervision of INERIS quality assurance. The dosing results may thus be considered as reliable. However, the analytical part of the current study was not GLP-Compliant, and the results are presented as informative only. The results obtained for the concentration assay of Copper Guanylurea nitrate (CuGUN) in treatment formulations used in the main assays were considered as satisfactory.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 489 (In Vivo Mammalian Alkaline Comet Assay)

Deviations:

not applicable

GLP compliance:

yes

Remarks:

Study realised according to OECD GD 489 (2014) and GLP but the determination of Copper Guanylurea nitrate (CuGUN) in treatment formulations was performed by a non-GLP-compliant laboratory (ESCOM, France) using a method.

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

copper(2+) bis(carbamimidoylurea) dinitrate

EC Number:

800-038-5

Cas Number:

1071838-81-7

Molecular formula:

Cu(C2H6N4O)2 (NO3)2

IUPAC Name:

copper(2+) bis(carbamimidoylurea) dinitrate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

IPL REGISTRATION NUMBER : 160309
BATCH NUMBER : 40, INERIS ref12AE617*
EXPIRY DATE : September 30, 2018
APPEARANCE : blue powder
PURITY : 98.4% / copper, nitrate, guanylurea
SALT / BASE RATIO : unknown
WATER CONTENT : unknown
CORRECTION FACTOR : 1.016**
QUANTITY SUPPLIED : 27.63, 26.21 and 27.27 g
STORAGE CONDITIONS : room temperature (+20±5°C), protected from light and from humidity:
with humidity less than 60%. Vapour pressure should be lower than
9x10
-7
at 20 °C and away from any oxidizers.
STABILITY UNDER
STORAGE CONDITIONS : in process (no change since 2012 as indicated in the shelf life letter
(October 12, 2015)). Up to September 30, 2018 for batch 40

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

selection issued from preliminary toxicity and main assays

Sex:

male

Details on test animals or test system and environmental conditions:

Young adult 8 weeks old male OFA Sprague-Dawley rats (Charles River France origin, Saint-Germain-sur-l’Arbresle; FRANCE) were used for the study. Body weight in male rats of the main assay ranged between 177 g and 210 g.
The period of acclimatization was of 6 day for the preliminary toxicity assay and for the main assay. It was of 7 days for the confirmatory toxicity assay. The animals received a clinical examination in order to retain only those which were healthy. The animals were identified by numbered ear rings.
The bedding consists of dust-free, irradiated softwood pellets.
The animals were dispatched in polypropylene cages by random-distribution.
The cages were placed in a ventilated system in the animal room, which was also ventilated. A timer provides lighting 12 hours a day (8 a.m. - 8 p.m.) in all the animal room. The temperature in the ventilated animal cupboard was 22 ± 3 °C, and humidity was 55 ± 15 %, with minor exceptions. The animals were not fasted at the treatment time. Drinking water, softened by reverse osmosis and filtered on 0.22 µm membrane, was provided ad libitum. The feedstuff used was A04C-10 from SAFE (batch 15230).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Copper Guanylurea nitrate (CuGUN) was suspended in drinking water (provided by IPL under the form of osmosed water). The stability of the test item in suspension in drinking water at 2 and 200 mg/mL is of 7 days at room temperature protected from light (CiToxLAB Study No. 39705 AHS).

Details on exposure:

For the preliminary toxicity assay, suspensions at initial concentration of 200, 125 and 80 mg/mL were prepared and administered to the animals at the dose volume of 10 mL/kg, giving final doses of 2000, 1250 and 800 mg/kg, respectively. For the 80 and 125 mg/mL initial concentrations, only one formulation was prepared and used for both treatments while a suspension at 200 mg/mL was extemporaneously made for each treatment of the 2000 mg/kg treatment group,.
For the confirmatory toxicity assay, a suspension at an initial concentration of 80 mg/mL was prepared and administered to the animals at the dose volume of 10 mL/kg, giving a final dose of 800 mg/kg. A single suspension was prepared and used for both treatments.
In the main genotoxicity assay, three suspensions of the test item at the initial concentrations of 80 - 40 and 20 mg/mL were prepared, giving final doses of 800 - 400 and 200 mg/kg, respectively when administered at 10 mL/kg. For each concentration, a single suspension was prepared and used for both treatments.

Duration of treatment / exposure:

PRELIMINARY TOXICITY ASSAY: 2 daily treatments at 24-hour interval
CONFIRMATION TOXICITY ASSAY: 2 daily treatments at 24-hour intervals
COMET ASSAY: 2 daily treatments at 24-hour intervals

Frequency of treatment:

PRELIMINARY TOXICITY ASSAY: 2000 – 1250 – 800 mg/kg/day (x 2)
CONFIRMATION TOXICITY ASSAY: 800 mg/kg/day (x 2)
COMET ASSAY: 800 – 400 – 200 mg/kg/day (x 2)

Post exposure period:

48 hours after 2nd treatment

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:

Basis:

Doses / concentrations 2

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Preliminary toxicity assay, 800 mg/kg/day (x 2)

Doses / concentrations 3

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Remarks:

Preliminary toxicity assay, 1250 mg/kg/day (x 2)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Preliminary toxicity assay, 2000 mg/kg/day (x 2)

Doses / concentrations 5

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Confirmatory toxicity assay, 800 mg/kg/day (x 2)

Doses / concentrations 6

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

COMET ASSAY, 200 mg/kg/day (x 2)

Doses / concentrations 7

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

COMET ASSAY, 400 mg/kg/day (x 2)

Doses / concentrations 8

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

COMET ASSAY, 800 mg/kg/day (x 2)

No. of animals per sex per dose:

PRELIMINARY TOXICITY ASSAY: 2 males
CONFIRMATION TOXICITY ASSAY: 5 males
COMET ASSAY: 5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Methylmethane sulfonate (MMS; Aldrich, batch MKBL6789V in sterile water, Fresenius, batch 13KAP031), 100 mg/kg (x2), oral administration at 24-hour interval

Examinations

Tissues and cell types examined:

Liver and glandular stomach

Details of tissue and slide preparation:

A portion of the liver and glandular stomach was removed (one part of each organ was preserved in formalin for eventual further analysis) and washed in the cold mincing buffer until as much blood as possible has been removed. The portion was minced with a pair of fine scissors to release the cells. The cell suspension was stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected.
Cells were enumerated on a haemocytometer, and sufficient cells to obtain 20 x 10-3 viable cells per slide were harvested from each cell suspension for proceeding to slides preparation.
A statistically significant increase in the occurrence of hedgehogs in liver cells was noted at the highest dose of 800 mg/kg/day (x2), i.e. slightly above the maximal value already noted in historical data for negative control (i.e. 5.59%, see Appendix No. 7), but no dose-response relationship was noted and the effect was thus not biologically relevant.
No statistically significant increase in the occurrence of hedgehogs in glandular stomach cells was noted at any of the doses tested when compared to the respective control.

Evaluation criteria:

A study is accepted if the following criteria are fulfilled:
- Concurrent negative controls should be within the control limits of laboratory’s historical negative control database.
- The positive controls should induce responses that are comparable to the historical positive control data and produce a statistically significant increase compared with the negative control.
- The appropriate number of doses and cells must be analysed.

Moreover:
- In the vehicle group, for each organ, an eventual increase in the frequency of hedgehogs, must not be >50%.
- If death(s) is(are) observed at the tested doses, the mortality rate must be less than 20 % per group.

The validity criteria for the test were considered as fulfilled and the test was validated.

For a test item to be considered positive in the comet assay, it must be observed:
- At least one of the treatment groups exhibits a statistically significant increase in the mean of medians of percentage of DNA in tail compared with the negative control,
- This increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
- Any of these results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce DNA strand breakage in the tissues studied in this test system.

A test item is considered clearly negative if:
- none of the test doses exhibits a statistically significant increase compared with the negative control,
- there is no dose-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical negative control data for a given species, vehicle, route, tissue, and number of administrations
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated.
The test chemical is then considered unable to induce DNA strand breakage in the tissues studied.

Statistics:

In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software Stat view®, version 5. As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian distribution (E. Bauer et al., 1998), the non-parametric, one-way Kruskall-Wallis test was performed. This method is based on the analysis of variance by ranks for testing equality of population medians among groups. The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the vehicle control in order to determine statistical significance of differences in group median values between each group versus the vehicle control. This test was also used to compare vehicle control and positive control to determine acceptable criteria of a valid test.

Results and discussion

Test results

Test results 1

Sex:

male

Genotoxicity:

negative

Remarks:

LIVER CELLS (200 mg/kg/day)

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

LIVER CELLS (400 mg/kg/day)

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

LIVER CELLS (800 mg/kg/day)

Toxicity:

yes

Remarks:

Diarrhea and slight decrease in spontaneous motor activity

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Sex:

male

Genotoxicity:

negative

Remarks:

GLANDULAR STOMACH CELLS (200 mg/kg/day)

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Sex:

male

Genotoxicity:

negative

Remarks:

GLANDULAR STOMACH CELLS (400 mg/kg/day)

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Sex:

male

Genotoxicity:

negative

Remarks:

GLANDULAR STOMACH CELLS (800 mg/kg/day)

Toxicity:

yes

Remarks:

Diarrhea and slight decrease in spontaneous motor activity

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

PRELIMINARY TOXICITY ASSAY

DOSES in mg/kg/day (x2)	DOSING VOLUME	NUMBER OF ANIMALS MALE RAT	% MORTALITY 24 HOURS AFTER FIRST TREATMENT	% MORTALITY 48 HOURS AFTER LAST TREATMENT
2000	10 mL/kg	2	0	50
1250	10 mL/kg	2	0	50
800	10 mL/kg	2	0	0

CONFIRMATORY TOXICITY ASSAY

DOSES in mg/kg/day (x2)	DOSING VOLUME	NUMBER OF ANIMALS MALE RAT	% MORTALITY 24 HOURS AFTER FIRST TREATMENT	% MORTALITY 48 HOURS AFTER LAST TREATMENT
800	10 mL/kg	5	0	0

COMET ASSAY IN LIVER CELLS

GROUP	TEST ITEM	DOSES in mg/kg/day (x2)	% of DNA in tail (mean of medians per animal (/5 animals))	NON PARAMETRIC statistical assessment (p Kruskall- Wallis)		NON PARAMETRIC statistical assessment (p Mann-Whitney)	Hedgehogs (Relative ratio of hedgehogs)	Hedgehogs (p)
Negative control	Drinking water	0	0.75	< 0.01		-	-	-
TREATED	CuGUN	800	10.45	< 0.01		< 0.01	1.68	< 0.05
TREATED	CuGUN	400	4.58	< 0.01		< 0.01	0.96	N.S.
TREATED	CuGUN	200	1.36	< 0.01		N.S.	1.15	N.S.
Positive control	Methylmethane sulfonate	100	48.35	-		< 0.01	1.03	N.S.

COMET ASSAY IN GLANDULAR STOMACH CELLS

GROUP	TEST ITEM	DOSES in mg/kg/day (x2)	% of DNA in tail (mean of medians per animal (/5 animals))	NON PARAMETRIC statistical assessment (p Kruskall- Wallis)		NON PARAMETRIC statistical assessment (p Mann-Whitney)	Hedgehogs (Relative ratio of hedgehogs)	Hedgehogs (p)
Negative control	Drinking water	0	22.38	< 0.01		-	-	-
TREATED	CuGUN	800	7.03	< 0.01		< 0.01	1.16	N.S.
TREATED	CuGUN	400	12.98	< 0.01		< 0.05	0.82	N.S.
TREATED	CuGUN	200	14.77	< 0.01		< 0.05	0.89	N.S.
Positive control	Methylmethane sulfonate	100	66.69	-		< 0.01	1.95	< 0.01

Applicant's summary and conclusion

Conclusions:

Under these experimental conditions, Copper Guanylurea nitrate (CuGUN) induced no statistically or biologically significant increases in DNA strand breaks at 800, 400 and 200 mg/kg/day (x2) in male rat isolated glandular stomach cells after oral administration. Therefore, Copper Guanylurea nitrate (CuGUN) is considered having no genotoxic activity in this organ.
 In return, in these conditions, Copper Guanylurea nitrate (CuGUN) induced dose-related statistically and biologically significant increases in DNA strand breaks at 800 and 400 mg/kg/day (x2) in male rat isolated liver cells after oral administration. Therefore, Copper Guanylurea nitrate (CuGUN) has been considered having a genotoxic activity in this organ.

Executive summary:

The test item Copper Guanylurea nitrate (CuGUN) (batch 40) provided by LIVBAG SAS was investigated by the means of the in vivo comet assay under alkaline conditions (SCGE) in the liver and glandular stomach of male rat treated orally twice with 800, 400 and 200 mg/kg/day, with one sampling time 2 to 6 hours after the last treatment according to OECD guideline (OECD 489, 2014). The highest dose was set according to the toxicity induced by the test item.

 The determination of Copper Guanylurea nitrate (CuGUN) in treatment formulations was performed by a non-GLP-compliant laboratory (ESCOM, France) using a method that was similar in its sensitivity for the dose level of copper to be assayed, to the one validated and used by the GLP-compliant laboratory INERIS. Analysis were performed with the presence of GLP-compliant INERIS study personnel and under the supervision of INERIS quality assurance. The dosing results may thus be considered as reliable. However, the analytical part of the current study was not GLP-Compliant, and the results are presented as informative only.

 The results obtained for the concentration assay of Copper Guanylurea nitrate (CuGUN) in treatment formulations used in the main assays were considered as satisfactory.

 The validity criteria for the study were met. The current study in both organs is thus considered as valid.

 Under these experimental conditions, Copper Guanylurea nitrate (CuGUN) induced no statistically or biologically significant increases in DNA strand breaks at 800, 400 and 200 mg/kg/day (x2) in male rat isolated glandular stomach cells after oral administration. Therefore, Copper Guanylurea nitrate (CuGUN) is considered having no genotoxic activity in this organ.

 In return, in these conditions, Copper Guanylurea nitrate (CuGUN) induced dose-related statistically and biologically significant increases in DNA strand breaks at 800 and 400 mg/kg/day (x2) in male rat isolated liver cells after oral administration. Therefore, Copper Guanylurea nitrate (CuGUN) has been considered having a genotoxic activity in this organ.