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

Endpoint summary

Description of key information

Oral repeated dose toxicity:
• No observed adverse effect at 200 mg/kg. Higher potassium level in females, albumin/globulin ratio in both sexes, minor histological changes, soiled urogenital, anal or buccal regions, squamous cell hyperplasia in the stomach were noted but not considered to be adverse.
• Target organs: haematology, liver, kidney, gastro-intestinal tract. 
• Clinical signs at 700/500 mg/kg: four treatment-related mortalities, lower body weight gains (associated with lower food consumption), hunched posture, piloerection, breathing difficulties,  soiled urogenital, anal or buccal regions, increase of abdomen size, higher platelets count and reticulocytes percentages, lower MCV and MCH, lower haemoglobin concentration, packed cell volume and prolonged mean prothrombin time, higher aspartate aminotransferase and alanine aminotransferase activities, higher potassium level and albumin/globulin ratio.
• Macroscopic adverse effect at 700/500 mg/kg: Squamous cell hyperplasia at the level of the limiting ridge in the stomach and dilatation/distension of various parts of the small or large intestine.
• Microscopic adverse effect at 700/500 mg/kg: hyaline droplets and tubular degeneration/regeneration in kidneys and single cell necrosis with inflammatory cell foci in the liver. In the gastro-intestinal tract, hyperplasia at the level of the limiting ridge, atrophy of the glandular portion of the stomach and hypertrophy or villous atrophy of the small intestine.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 5 weeks
- Weight at study initiation: The males had a mean body weight of 178 g (range: 165 g to 190 g) and the females had a mean body weight of 159 g (range: 150 g to 172 g)
- Housing: The animals were housed by five, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm2) containing autoclaved sawdust (SICSA, Alfortville, France). Each cage contained enrichment (rat hut).
- Diet (e.g. ad libitum): All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 2537604 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 2012-12-26 To: 2013-01-23

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Based on the recommendation of the Sponsor and on the validation of analytical method CiToxLAB France/Study No. 39704 VAA, the vehicle was drinking water treated by reverse osmosis using ELIX 5 (Millipore SA).

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a homogenous suspension in water.
The test item was mixed with the required quantity of water, under magnetic stirring, in order to obtain the desired concentrations.
The dose formulations were kept under magnetic stirring for at least 30 minutes. The frequency of dose formulation preparation was based on available stability data (up to 7 days). The dose formulations were stored at room temperature and protected from light, and delivered to the study room in brown flasks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The ion-exchange chromatography with UV detection analytical method for the determination of Copper Guanylurea Nitrate in dose formulation samples was provided by the Sponsor and the validation of the analytical method was conducted in CiToxLAB France/Study No. 39704 VAA. Precise details concerning the checked parameters, acceptance criteria and obtained results are documented in the corresponding validation report.

Before the start of treatment the suitability of the dose formulation process was confirmed. The homogeneity and stability were also determined on a range of dose formulations prepared at levels which cover the lowest and highest concentrations proposed for use in this study.
Precise details concerning the validation process, the homogeneity and stability study are documented in the CiToxLAB France/Study No. 39705 AHS.

The concentration of the test item in samples of each control and test item dose formulation prepared for use in weeks 1, 2, 3 and 4 was determined. Analysis of dose formulation prepared at 100 mg/mL was performed twice during the study.

The test item concentrations in the administered dose formulations analyzed in weeks 1, 2, 3 and 4 remained within an acceptable range of -5.5% to +3.5% when compared to the nominal values (± 15%).

Duration of treatment / exposure:

28 days (day 1 is the first day of treatment)

Frequency of treatment:

every day

Remarks:

Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
200 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
700/500 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected based on the results of CiToxLAB France/Study No. 39681 TSR (the 14-day range finding toxicity study by oral route in rats). In this study, three groups of three males and three females received the test item by gavage at 100, 300 or 1000 mg/kg/day as a solution in drinking water and a group of three males and three females received the vehicle only. Animals given 1000 mg/kg were treated until day 3 and were prematurely killed or found dead on days 4, 5 or 6. Another group of one male and one female was added and animals received 600 mg/kg during 9 days. There were no obvious test item-related effects in animals receiving 300 and 600 mg/kg/day.
Therefore, 700 mg/kg/day was selected as the high dose-level for this 4-week study. Mid and low dose-levels were selected in order to cover approximately 3- to 4-fold intervals.

- Administration:
The dose formulations were administered by gavage, using a plastic syringe fitted with a metal gavage tube, once a day at approximately the same time. The quantity of dose formulation administered to each animal was adjusted according to the most recently recorded body weight. A constant dosage-volume of 5 mL/kg/day was used, except for group 4 on days 9 and 10 which was treated with the dosage-volume of 3.57 mL/kg/day from the 140 mg/mL formulation. Control animals (group 1) received the vehicle only. The dose formulations were stirred continuously throughout the dosing procedure.

- Rationale for animal assignment (if not random): The animals were allocated to groups (by sex) using a computerized stratification procedure so that the average body weight of each group was similar

Positive control:

no positive control

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was observed once a day, at approximately the same time, for the recording of clinical signs.
Male A25980 (group 2) presenting a wound in the dorsal region from day 16 was given Cothivet twice a day, on days 21 to 28.
Male A25989 (group 4) presenting abdomen increased in size, received 5 mL/kg of paraffin oil on day 6.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then once a week until the end of the study
- Detailed clinical observations: included (but were not limited to) changes in the skin, fur, eyes and mucous membranes, occurence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) and bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded

BODY WEIGHT: Yes
- Time schedule for examinations: once before group allocation, on the first day of treatment, at least once a week until the end of the study.
In week 2, high dose-level group was weighed twice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Time schedule for examinations: once a week in each cage until the end of the study.
Food consumption was calculated per animal and per day.
When one of the animals in the same cage died, the number of days for which that animal had been present in the cage was taken into consideration for the calculation of food consumption.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION : No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, for an overnight period of at least 14 hours before blood sampling.
- How many animals: all surviving animals
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Animals fasted: Yes, for an overnight period of at least 14 hours before blood sampling
- How many animals: all surviving animals
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for an overnight period of at least 14 hours before urine collection
- Parameters checked in table [No.2] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once in week 4
- Dose groups that were examined: alls animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
- Detailed clinical observations: the following parameters were assessed and graded:
 in the cage: "touch escape",
 in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
 in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.
- Reactivity to manipulation and different stimuli: the following parameters measurements, reflexes and responses were recorded:
 touch response,
 forelimb grip strength,
 pupillary reflex,
 visual stimulus response,
 auditory startle reflex,
 tail pinch response,
 righting reflex,
 landing foot splay,
 rectal temperature (at the end of the observation period).
- Motor activity: motor activity was measured by automated infra-red sensor equipment over a 60 minute period.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (table no.3)
HISTOPATHOLOGY: Yes (table no.3)

Statistics:

citox software (version D.6) was used to perform the statistical analyses of body weiht, hematology, clinicla chemistry and urinalysis data according to a sequence attached as background material.
Pathdata software (version 6.2d2) was used to perform the statistical analysis of organ weight data according to the sequence attached as background material.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Four animals died during the study. 1 male and one female treated at 700 mg/kg/day were prematurely sacrificed on day 8. One male treated at 700 mg/kg/day and one female treated at 700 and then 500 mg/kg/day were found dead on days 7 and 21, respectively.

Mortality:

mortality observed, treatment-related

Description (incidence):

Four animals died during the study. 1 male and one female treated at 700 mg/kg/day were prematurely sacrificed on day 8. One male treated at 700 mg/kg/day and one female treated at 700 and then 500 mg/kg/day were found dead on days 7 and 21, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 700/500 mg/kg/day, lower mean body weight gain was observed in both sexes during the treatment period in high-dose animals, triggering lower mean body weight at the end of treatment period, when compared to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Compared to controls, marked lower mean food observed in males treated at 700 mg/kg/day during days 1 to 7, days 8 to 14 and days 22 to 27. Lower mean food consumption noted in females treated at 700 mg/kg/day in the first and third weeks of treatment.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

lower hemoglobin concentration and packed cell volume observed in males treated at 700/500 mg/kg/day. Marked to severe higher platelet seen in both sexes. Higher reticulocyte % seen in males.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Minor toxicological importance

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

decrease in mean pH in males treated at 700 mg/kg/day.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

test item-related effect on testes and epididymides

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

test item-related changes observed at 200 and 700/500 mg/kg/day mainly in the gastro-intestinal tract.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No test item-related changes

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

kidney changes at 700/500 mg/kg/day were considered to be adverse. Liver: changes considered to be fortuitous and without relationship with the test item. Forestomach and stomach: effect not considered to be adverse.

Details on results:

CLINICAL SIGNS AND MORTALITY
Four animals died during the study, as follow:
- one male (A25987) and one female (A26029) treated at 700 mg/kg/day were prematurely sacrificed on day 8,
- one male (A25989) treated at 700 mg/kg/day and one female (A26027) treated at 700 and then 500 mg/kg/day were found dead on days 7 and 21, respectively.



BODY WEIGHT AND WEIGHT GAIN

HAEMATOLOGY

CLINICAL CHEMISTRY

URINALYSIS

NEUROBEHAVIOUR

ORGAN WEIGHTS

GROSS PATHOLOGY

HISTOPATHOLOGY: NON-NEOPLASTIC

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) was established at 200 mg/kg/day and the No Observed Effect Level (NOEL) at 50 mg/kg/day

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item, Copper Guanylurea Nitrate following daily oral administration (gavage) to rats for 4 weeks.

Three groups of five male and five female Sprague-Dawley rats received the test item, Copper Guanylurea Nitrate (batch No. 40), by daily oral administration for 28 days, at dose-levels of 50, 200 or 700/500 mg/kg/day (700 mg/kg/day from day 1 to day 8 and 500 mg/kg/day from day 9 until the end of study). The test item was administered as a suspension in the vehicle (drinking water treated by reverse osmosis) at a constant dosage-volume of 5 mL/kg/day, except for group 4 on days 9 and 10 which was treated with the dosage-volume of 3.57 mL/kg/day from the 140 mg/mL formulation. A control group of five males and five females received the vehicle alone under the same experimental conditions. The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. In addition, detailed clinical examinations were performed once weekly. Body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as food consumption. Towards the end of the dosing period, a Functional Observation Battery including motor activity measurement, and hematology, blood biochemistry and urinalysis were performed on all surviving animals. On completion of the treatment period, the animals were euthanized and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on selected tissues from control- and high-dose animals and all macroscopic lesions from all low- and intermediate-dose animals.

The test item concentrations in the administered dose formulations analyzed in weeks 1, 2, 3 and 4 were within an acceptable range when compared to the nominal values.

Four animals died during the study: one male and one female treated at 700 mg/kg/day were prematurely sacrificed on day 8 and one male treated at 700 mg/kg/day and one female treated at 700/500 mg/kg/day were found dead on days 7 and 21, respectively. At microscopic examination, test item-related changes were noted in kidneys, liver and gastro-intestinal tract. All these deaths were considered to be related to treatment with the test item.

In animals treated at 700/500 mg/kg/day, clinical signs indicative of poor clinical condition were observed in some surviving animals, notably thin appearance, hunched posture, piloerection and breathing difficulties. Other clinical signs, like soiled urogenital anal, or buccal regions, increase in size of abdomen were observed. Clinical signs described above were considered as adverse as they had marked consequences on body weight gains, food consumption, hematology and blood biochemistry parameters and general health of the animals. In animals treated at 200 mg/kg/day, soiled urogenital, anal or buccal regions were noted.

At motor activity analysis, there were no relevant differences in rearing and horizontal movements in test item-treated groups in both sexes when compared to control group.

At 700/500 mg/kg/day, lower mean body weight gain was observed in both sexes during the treatment period in high-dose animals, triggering lower mean body weight at the end of treatment period, when compared to controls.

Lower food consumption was recorded in the high-dose males and females during the first week of treatment period, when compared to controls. It was correlated with the effects on mean body weight.

In animals treated at 700/500 mg/kg/day, lower hemoglobin concentration and packed cell volume associated prolonged prothrombin time were observed in males, when compared to controls. In both sexes, lower Mean Cell Volume (MCV) and Mean Cell Hemoglobin (MCH) with marked higher platelet counts and higher reticulocyte percentage and were observed.

At blood biochemistry analysis, higher aspartate aminotransferase and alanine aminotransferase activities were observed in both sexes treated at 700/500 mg/kg/day, when compared to control group. In addition, higher potassium level and higher albumin/globulin ratio were noted in females.

In animals treated at 200 mg/kg/day, higher potassium level was noted in females when compared to control group.

At urinalysis, the only statistically significant difference was noted in the pH of the high-dose males.

At macroscopic evaluation, squamous cell hyperplasia at the level of the limiting ridge was seen in the stomach and dilatation/distension of various parts of the small or large intestine were observed in animals treated at 200 and 500/700 mg/kg/day. At microscopic analysis, test item related changes observed in animals given 700/500 mg/kg were seen in kidneys, liver and gastro-intestinal tract. In kidneys, hyaline droplets and tubular degeneration/regeneration were observed in kidneys of both sexes. In the liver, single cell necrosis with inflammatory cell foci were present. In the gastro-intestinal tract, changes involved the forestomach (hyperplasia at the level of the limiting ridge often associated with hyperkeratosis correlating with white discoloration and thickening at necropsy), the stomach (minimal atrophy of the glandular portion) and small intestine (hypertrophy or villous atrophy). Non specific changes, considered to be stress related included lymphoid atrophy (thymus, spleen, mesenteric lymph nodes), decreased content in prostate/seminal vesicles and higher adrenal, weight.

At 200 mg/kg/day changes were non adverse and seen at a low severity. They included minimal hyaline droplets in kidneys, minimal to slight hyperplasia, occasionally associated with hyperkeratosis in the forestomach (correlating with thickening and white discoloration at necropsy) and minimal atrophy of the glandular mucosa in the stomach in one male and one female.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a sub-chronic toxicity study (90 days) does not need to be conducted because a reliable short-term toxicity study (28 days) is available showing severe toxicity effects according to the relevant criteria for classifying the substance, for which the observed NOAEL-28 days, with the application of an appropriate uncertainty factor, allows the extrapolation towards the NOAEL-90 days for the same route of exposure

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Only one study available but quality is sufficient for effect level determination.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The tubular degeneration/regeneration in kidneys and single cell necrosis with inflammatory cell foci in the liver observed in rats (males and females) exposed to 500 and 700 mg/kg/day of CuGUN is probably relevant to humans. These effects are observed for the two highest doses and are taken into account in risk assessment. At 200 mg/kg/day, no adverse effect was observed.

No data are available for other routes of exposure (inhalation and dermal route). However, potential effects during repeated exposures to CUGUN are taken into account for risk assessment (route to route extrapolation) even if CuGUN is not classified for these routes of exposure. The exposure by inhalation is expected to be low based on the very low vapour pressure (0.0000009 Pa ) and the particles size (only 10 % of particles are below 13.5 µM (more than 90 % of particles are non inhalable)). Moreover, according to the acute toxicity study results, CuGUN seems to be more toxic by oral route than dermal route. This is probably due to a more important absorption by oral route than dermal.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study was performed in accordance to OECD 407 and EC B.7 guideline, and to GLP. No deviation was observed.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: other; digestive: liver; digestive: stomach; urogenital: kidneys

Justification for classification or non-classification

According the equivalent guidance values for 28-day study of ECHA guidance document (Guidance on the application of the CLP criteria, version 4.0, November 2013) and the 1272/2008 regulation (CLP), CuGUN is classified as "Specific target organ toxicity - repeated exposure, category 2", based on a NOAEL of 200 mg/kg/day (which is included in the category 2 values: 30 - 300 mg/kg/day).