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

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05/10/12 to 10/02/12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

study performed in accordance to OECD guideline 423 and GLP except for the analysis of CuGUN. Analysis of CuGUN was performed by the chemical analysis unit of INERIS wich is not included in the GLP area of expertise of the test facility. However, this analytical phase was performed according to INERIS recorded and validated procedures, and was inspected by the Quality Assurance Personnel of the test facility for both the technical phase and the raw data generated. As a consequence, due to the approach used for control of these data, it was considered that the non-GLP analytical monitoring of the test item did not affect the study compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

yes

Remarks:

some temperature and hygrometry deviations were observed. Based on animal clinical observation, the study director concluded these deviations did not have any significant impact on the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

except analysis of the test item (see rationale for reliability)

Test type:

acute toxic class method

Limit test:

no

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

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 230g (group 300mg/kg) and 236g (group 2000mg/kg)
- Fasting period before study: approximately 5:00 pm on day before treatment to 3 hours after treatment
- Housing: in polycarbonate cages containing dust-free bedding (sawdust + chips), cages were changed at least once a week
- Diet (e.g. ad libitum): ad libitum except during fasting period, rat/mice altromin 1320, Genestil
- Water (e.g. ad libitum): ad libitum, filtered (0.2 µm) tap water
- Acclimation period: 5 to 19 depending on treatment session

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 45% to 65%
- Air changes (per hr): 15 to 20 cycles
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 2012-5-16 (first exposure) To: 2012-6-07 (last necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

filtered tap water

Details on oral exposure:

The vehicle used in the study was selected from the results of solubility and homogeneity assays performed prior to the main test. A homogeneous suspension was obtained with filtered tap water at concentrations of 30 g/L and of 200 mg/L of CuGUN

MAXIMUM DOSE VOLUME APPLIED: 10ml/kg (max 2.4 ml /boby weight 240g)

DOSAGE PREPARATION (if unusual): CuGUN preparations, administered as a homogeneous suspension, were prepared approximately within 1h00 of dosing. CuGUN was weighted and diluted with the filtered tap water volume to obtain the desired concentration. CuGUN preparations were stirred continuously, in order to maintain a homogeneous suspension, throughout the administration period.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose:
the initial dose of 300mg/kg was chosen in accordance wit the OECD No.423 guideline
a higher dose (2000mg/kg) was subsequently selected based on the absence of mortality observed in the rats treated with the initial dose

Doses:

Animals were treated at the following doses: 297 ± 4 mg/kg and 1,996 ± 21 mg/kg

No. of animals per sex per dose:

6

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: once on the day of arrival, once daily during the acclimation period except during the week-end, several times on the day of treatment (D0): at least once during the first 30 minutes after administration, at least 2 times during the following 4 hours, and at least once thereafter, daily from D1 to D14 (once or twice per day).
- Frequency of weighing: once on the day of arrival, once on the day of selection, once on day -1 (before fasting period), once on day 0, before treatment, once daily from D1 to D14.
- Necropsy of surviving animals performed: yes
- Other examinations performed: clinical signs, body weight, macroscopic observation of thoracic area organs (respiratory tracts (trachea, lungs), the tracheo-bronchial lymph nodes, heart, and oesophagus without opening of the tube) and abdominal organs (liver, spleen, pancreas, digestive tract (without opening of the tube, except stomach), adrenal glands, kidneys, bladder, liver and kidneys lymphatic ganglia, lumbar aortic lymphatic ganglia and female reproductive organs), histopathology of organs with macroscopic abnormalities

Statistics:

Statistical analysis was performed on body weight data (body weight and body weight change) using the GraphPad Prism® software. Assuming that body weight data are normally distributed, an ANOVA test was applied and a Bonferroni's posthoc analysis was chosen to compare daily body weight means between group 2 and group 3.

Results and discussion

Effect levels

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - < 2 000 mg/kg bw

Mortality:

2 rats, treated with 2,000 mg/kg, were found dead at day 1 (1 rat) and at day 3 (1 rat). 2 rats, treated with 2,000 mg/kg, were euthanized because they presented signs of suffering and important weight loss approximately 20% of total weight measured before the treatment.

Clinical signs:

other: No clinical signs were observed on rats treated with the test item at 300 mg/kg, during the observation period. All rats treated with the test item at 2,000 mg/kg, presented an excessive salivation, for 10 or 30 minutes after treatment. The rat treat

Gross pathology:

Macroscopic changes considered to be related to the treatment of CuGUN were observed by external examination and in stomach, in small and large intestine, in rectum and in kidneys in rats treated with 2,000 mg/kg dose which died before the end of the study. Brown nose (2 rats), brown contour of the eyes (1 rat), soiled urogenital area (1 rat) and soiled blue green anogenital area (1 rat) were observed by external examination. Stomach presented mucosal red spots/areas (4 rats), was distended (1 rat), was filled with dark or black/blue colour content (3 rats). Small and large intestine were normal but empty of faecal matter (2 rats) and filled with black mucous (1 rat) or gray liquid content (1 rat). Some black faeces were found in rectum of one rat. Kidneys presented some dark spots (1 rat).

Other findings:

Microscopic changes considered to be related to treatment were restricted to stomach (3 rats), forestomach (1 rat), kidneys (1 rat) and thymus (1 rat) from rats treated with 2,000 mg/kg of CuGUN which died before the end of the study. It consisted in degeneration/necrosis/apoptosis in the stomach/forestomach, lymphocytolysis in the thymus and tubular degeneration/regeneration with crystal deposition in the kidneys.

Any other information on results incl. tables

Dose preparation:

Date (YYYY-mm-dd)	test item mass	total mass of the preparation	concentration	animal group
2012 -05 -16	3.00 g	100.00 g	30.0 g/L	2 (step 1)
2012 -05 -21	3.01 g	100.01 g	30.1 g/L	2 (step 2)
2012 -05 -24	20.00 g	101.13 g	200.0 g/L	3 (step 1)
2012 -05 -30	20.01 g	100.01 g	200.1 g/L	3 (step 2)

Dose administration, mortality and clinical signs: individual data

animal group	animal	Body weight at day 0	administered volume	Test item concentration	administered dose	mortality	clinical signs
2 (step 1)	161	210.7 g	2.1 ml	30.0 g/L	299.0 mg/L	scheduled death at day 14	no clinical signs observed
2 (step 1)	162	203.4 g	2.0 ml	30.0 g/L	295.0 mg/L	scheduled death at day 14	no clinical signs observed
2 (step 1)	163	213.1 g	2.1 ml	30.0 g/L	295.6 mg/L	scheduled death at day 14	no clinical signs observed
2 (step 2)	164	225.9 g	2.2 ml	30.1 g/L	292.2 mg/L	scheduled death at day 14	no clinical signs observed
2 (step 2)	165	220.5 g	2.2 ml	30.1 g/L	299.4 mg/L	scheduled death at day 14	no clinical signs observed
2 (step 2)	166	217.9 g	2.2 ml	30.1 g/L	303.0 mg/L	scheduled death at day 14	no clinical signs observed
3 (step 1)	167	221.6 g	2.2 ml	200.0 g/L	1,985.6 mg/L	found dead at day 1	excessive salivation during 10 minutes after treatment
3 (step 1)	168	210.9 g	2.1 ml	200.0 g/L	1,991.5 mg/L	scheduled death at day 14	-excessive salivation during 30 minutes after treatment-digestive disorder: soft and blue faeces
3 (step 1)	169	228.0 g	2.3 ml	200.0 g/L	2,017.6 mg/L	scheduled death at day 14	-excessive salivation during 30 minutes after treatment-digestive disorder: soft and blue faeces
3 (step 2)	170	224.2 g	2.2 ml	200.1 g/L	1,963.0 mg/L	euthanized at day 4	-excessive salivation during 30 minutes after treatment-digestive disorder: absence of faeces-suffering signs: eyelids partially closed, piloerection, round back, loss of weight
3 (step 2)	171	228.0 g	2.3 ml	200.1 g/L	2,072.1 mg/L	euthanized at day 4	-excessive salivation during 30 minutes after treatment -digestive disorder: liquid and blue faeces-suffering signs: eyelids partially closed, piloerection, round back
3 (step 2)	172	240.2 g	2.4 ml	200.1 g/L	1,998.8 mg/L	found dead at day 3	-excessive salivation during 30 minutes after treatment -digestive disorder: liquid blue faeces, soiled area: anus blue and green-suffering signs: eyelids partially closed, piloerection, round back, loss of weight

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Remarks:

Migrated information

Conclusions:

According to these results, the classification (1272/2008/ECC regulation) of the test item, Copper Guanylurea Nitrate (CuGUN), is the following:
- Category 4; H302: harmful if swallowed.

Executive summary:

The objective of this study was to evaluate the acute oral toxicity of the test item Copper Guanylurea Nitrate (CuGUN), in rat. The study design was based on OECD Guideline No. 423 "Acute Oral Toxicity – Acute Toxic Class Method", December 17th, 2001.

Method

In the preliminary assay, the solubility, the stability and the homogeneity of the test item in filtered tap water were assessed at 30 g/L and 200 g/L. CuGUN preparations were stirred continuously, in order to obtain and maintain a homogeneous suspension, throughout the administration.

For each dose, six nulliparous and non gravid female WISTAR rats were treated with test item in 2 successive sessions (3 rats per session). The start of the next session and/or session using the higher dose of the test item were only carried out when the certainty was obtained that the rats, treated on the preceding session, survived.

For each session, each animal was observed at least once a day for mortality, clinical signs and body weight, during 15 days. On completion of the observation period, a necropsy including macroscopic examination was realized on animals after scheduled or unscheduled death, when possible. A microscopic examination was performed on organs with macroscopic abnormality.

Results

Test item doses and treatment

At 30 g/L and 200 g/L, the test item was not soluble in water; a homogeneous suspension was obtained in filtered tap water, which was stable for at least one hour, Animals were treated at the following doses: 297 ± 4 mg/kg and 1,996 ± 21 mg/kg.

Mortality and clinical signs:

No unscheduled deaths and no clinical signs were observed on rats treated with the test item at 300 mg/kg, during the observation period. All rats treated with the test item at 2,000 mg/kg, presented an excessive salivation, for 10 or 30 minutes after treatment. Unscheduled deaths of 4 rats (1 rat in the first session and 3 rats in the second session) treated at 2,000 mg/kg were noted between day 1 and day 4. Signs of toxicity were observed until their death: a weight loss, blue liquid faeces or absence of faeces, round back, piloerection, eyelids partially closed. The two rats treated at 2,000 mg/kg, which survived during the observation period, presented blue soft faeces on day 1 and day 2.

Macroscopic and microscopic examination

Macroscopic changes considered to be related to the treatment of CuGUN were observed by external examination and in stomach, in small and large intestine, in rectum and in kidneys in rats treated with 2,000 mg/kg dose which died before the end of the study. Brown nose (2 rats), brown contour of the eyes (1 rat), soiled urogenital area (1 rat) and soiled blue green anogenital area (1 rat) were observed by external examination. Stomach presented mucosal red spots/areas (4 rats), was distended (1 rat), was filled with dark or black/blue colour content (3 rats). Small and large intestine were normal but empty of faecal matter (2 rats) and filled with black mucous (1 rat) or gray liquid content (1 rat). Some black faeces were found in rectum of one rat. Kidneys presented some dark spots (1 rat).

Microscopic changes considered to be related to treatment were restricted to stomach (3 rats), forestomach (1 rat), kidneys (1 rat) and thymus (1 rat) from rats treated with 2,000 mg/kg of CuGUN which died before the end of the study. It consisted in degeneration/necrosis/apoptosis in the stomach/forestomach, lymphocytolysis in the thymus and tubular degeneration/regeneration with crystal deposition in the kidneys.

Conclusion

According to these results, the classification (1272/2008/ECC regulation) of the test item, Copper Guanylurea Nitrate (CuGUN), is the following:

-                   Category 4; H302: harmful if swallowed.