Copper, diammine[5,5'-bi-1H-tetrazolato(2-)-kN1, kN1']

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

14-day dose-range finding toxicity study by oral route in Wistar rats

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March/April 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Some minor deviations as aslo observed but conidered have no significant impact on the result and the validity of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

14-day dose-range finding toxicity study by oral route in Wistar rats. Some minor deviations as aslo observed but conidered have no significant impact on the result and the validity of the study.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Copper, diammine[5,5'-bi-1H-tetrazolato(2-)-kN1, kN1']

EC Number:

611-056-6

Cas Number:

538313-26-7

Molecular formula:

C2H6CuN10

IUPAC Name:

Copper, diammine[5,5'-bi-1H-tetrazolato(2-)-kN1, kN1']

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Provider : AUTOLIV ASP, 16700 W. Hwy 83, Promontory, UTAH 84307, USA
- batch No. P3656461
- Expiration date of the lot/batch: august 25th 2020
- Purity: 97.4%

STORAGE CONDITIONS OF TEST MATERIAL
Temperature and humidity restrictions have not been set. Warehouse recommendation of the test item in a depository designed for explosives has been accepted by AUTOLIV.
Temperature was recorded by a thermometer checked and calibrated according to standard operating procedures. From test item reception to the end of the in-life study, temperatures were comprised between -4.2 and 33.3°C in the premises dedicated.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar RjHan:WI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:JANVIER LABS
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 12 weeks
- Weight at study initiation: 460-465g (males), 240-250g (females)
- Fasting period before study:
- Housing: Animals were initially housed 1 or 2 per cage, and then individually the day before the beginning of the treatment.
Animals were housed in polysulfone cages (type 3 high, Tecniplast, France) with stainless steel lids (Tecniplast, France), containing autoclaved bedding (mix of corn and wood chips, MK2000 and Tierwohl, Genestil, France) and water bottles. They were changed once a week.
Each cage was labeled with the study number, the experimental group number, the type of treatment, the cage number and the animals' identification (number and sex).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: An acclimation period of 5 days for the animals treated with the high dose of CuDABT, 1000 mg/kg/day (G4), and controls (G1), and of 6 days for the animals treated with the low and the mid doses of CuDABT, respectively 100 and 300 mg/kg/day (G2 and G3), was respected before experiments. A delay in the start of the treatment was planned to perform the necropsies over 2 days.

DETAILS OF FOOD AND WATER QUALITY:
Animals had ad libitum access to food (Altromin, Genestil, France) and tap water (filtered with a 0.2 µm filter, Opticap® capsules with Milligard® media, Millipore) contained in polysulfone bottles supplied with stainless teats (Tecniplast, France). Pellets were removed before sacrifice of animals subjected to biochemical analyses (G1 and G4), on D15.
The diet batch was analyzed by Altromin Spezialfutter GmbH & Co. KG for composition and contaminant levels. The bedding batches were analyzed by NSF Erdmann Analytics GmbH for chemical and biological contaminants. The bacterial analysis of water, performed by the laboratory Wessling, showed the absence of bacteria at the detection level. All contaminant analyses certificates are joined to the study files.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 40 to 70%
Temperature and relative humidity were recorded continuously by a thermo-hygrograph, checked and calibrated according to internal standard operating procedures. These parameters were daily checked and reported in the study file. Monitoring data of artificial lighting were reported in the facility's files.
- Air changes (per hr): 10 to 20 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/24h

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral way has been selected since it’s a route of exposure regulatory requested for the toxicological evaluation of such type of test item.

Vehicle:

water

Remarks:

Milli-Q water

Details on oral exposure:

The dose administrated to animals corresponded to the ratio of the mass of the test item per kilogram of body weight, divided by the volume of solution per kilogram of body weight.
The dose formulations have been administered daily for a 14-day period by gavage (using a plastic syringe fitted with a rigid stainless cannula). Solutions were continuously stirred throughout dosing and the desired volume always removed from the center of the flask.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the test item preparations were assessed by the measurement of total copper concentration.
Copper was analyzed by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) after acid digestion of the samples. The copper analysis was performed in compliance with INERIS’ procedures and the NF EN ISO 11885 (11/2009) standard.
The samples preparation was based on a digestion procedure with addition of nitric acid (HNO3 69%) to obtain a 4% HNO3 solution. The solutions were shaken to increase the solubilization of the samples in acid conditions in a digestion tube. Final volume was adjusted at 50 mL with Milli-Q water before analysis.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Once a day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group G2

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group G3

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group G4

No. of animals per sex per dose:

3 males and 3 females

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Their health status was assessed by clinical observation on the day of arrival at the facility, and daily during the acclimation period. To be identified during this phase, animals were marked on the tail by one or two lines.
Each animal was checked for mortality and morbidity at least once a day.
Each animal has been subjected to general clinical observations at least once a day. Clinical sign(s) time of appearance, duration and time of disappearance have been noted when applicable.
The body weight of each animal was recorded the day of arrival, the day of randomization, before the beginning of the treatment period, on the 1st day of treatment, at least once a week until the end of the study, and before euthanasia.
The quantity of food consumed by each animal has been recorded once a week all along the study

Sacrifice and pathology:

On completion of the treatment period, all surviving animals have been weighted and euthanatized (D15) by an intraperitoneal injection of sodium pentobarbital (100 mg/kg of body weight).
A complete macroscopic post-mortem examination has been performed on all animals. This included the observation of the external surfaces, all orifices, the external surfaces of the brain, the cranial, thoracic, abdominal and pelvic cavities, and the neck with their associated organs and tissues.

Other examinations:

The quantity of food consumed by each animal has been recorded once a week all along the study.

Blood collection:
Prior to sacrifice, control animals and rats treated with the highest dose of CuDABT have been food deprived for at least 6 hours. After euthanasia, their blood has been collected at the vena cava into tubes containing an anticoagulant (EDTA tubes for hematology and lithium heparin tubes for biochemistry).

Hematology:
The following parameters have been determined using a hematology analyzer: hematocrit, hemoglobin concentrations, erythrocytes and reticulocytes count, total and differential leucocytes count, as well as platelet count.

Biochemistry:
A serum chemistry analyzer has been used to measure the following parameters: sodium, potassium, chloride, total CO2, Na/K, urea, creatinine, urea/creatinine ratio, creatinine kinase, glucose, total protein, albumin, gamma-GT, albumin/globulin ratio, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, amylase, total cholesterol, total bilirubin, calcium, phosphorus, and uric acid.

Statistics:

Effects of the test item were analyzed for each sex by mean of a Kruskall-Wallis test with the corresponding control group used as the reference. Results obtained from the hematological and biochemical assays were analyzed using a Student t-test. Statistical analysis was carried out by mean of SPSS Statistics 19 for Windows (SPSS Inc., Chicago, IL, USA). The differences were considered significant at the level of p < 0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical defects related of health degradation were observed in the two animals sacrificed before the end of the study:
-female No. 300 treated with 100 mg/kg/day: pilorection, hunched posture, eyelids partially closed, dyspnea, pale mucous and a decrease of activity from D12,
-female No. 302 treated with 300 mg/kg/day: pilorection, eyelids partially closed, noisy respiration, prostration, scab and a decrease of activity from D8.

Otherwise, difficulties of gavage were encountered and regurgitations were observed from few days after the 1st administration until the end of the treatment period. In fact, numerous rats regurgitated parts of the solution administered (2 females from the control group from D6, 3 females treated with 100 mg/kg/day from D7, 2 females treated with 300 mg/kg/day from D6 and 1 male and 1 female treated with 1000 mg/kg/day from D4). The volume administered, 10 mL/kg, has been suspected to be too important considering a daily administration on a repeated period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two females (No. 300 and 302, respectively treated with CuDABT at 100 and 300 mg/kg/day) were sacrificed before the end of the study because of health worsening (respectively at D12 and D10). Clinical signs of deteriorated welfare and severe pain having appeared rapidly, the Study Director decided to euthanised these animals.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant difference was observed in the body weight and body weight gain between the four groups all along the study period.
A decrease in body weight means was however noted in males treated with CuDABT at 1000 mg/kg/day from D2 to D15 compared to controls. The variability of the data being important in this group, no difference has been statistically highlighted. No effect of the treatment can thus be reported.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant difference was observed between the four groups concerning the mean daily quantity of food consumed, whatever the period considered (D1 to D7, D7 to D13 and D13 to D15).

Food efficiency:

no effects observed

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

No difference between control values and levels measured in blood of animals treated with the highest dose was revealed by the statistical analysis concerning hematocrit, hemoglobin concentrations, erythrocytes and reticulocytes count, total and differential leucocytes count, and platelet count.
However, inter-individual variability has been observed for some parameters in control and treated animals. These differences concerning only certain parameters, they do not reflect a biologically significant effect.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No difference between control values and levels measured in plasma of animals treated with the highest dose was revealed by the statistical analysis concerning sodium, potassium, chloride, total CO2, Na/K, urea, creatinine, urea/creatinine ratio, creatinine kinase, glucose, total protein, albumin, gamma-GT, albumin/globulin ratio, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, amylase, total cholesterol, total bilirubin, calcium, phosphorus, and uric acid.
Inter-individual variability has also been observed for some biochemical parameters in control and treated rats. Since these differences concerned only certain parameters, they do not reflect either a biologically significant effect.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

In the two animals sacrificed before the end of the study, the following internal abnormalities have been observed:
- female No. 300 treated with 100 mg/kg/day: perforation of the esophagus,
- female No. 302 treated with 300 mg/kg/day: lungs of abnormal colour and size, trachea of abnormal colour.

These macroscopic observations lead to suppose that clinical effects were not related to a toxicity of the test item, but rather on technical aspects as gavage defects.
In two animals euthanised at the end of the study, the following internal macroscopic abnormalities have been observed:
- male No. 281 from the control group: presence of a pancreatic node, and liver of abnormal size and colour,
- male No. 284 treated with 1000 mg/kg/day: lungs of abnormal colour.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Effect levels

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The oral exposure to CuDABT during 14 days induced no clinical disruptions, as well as no changes in body weight and food consumption in adult male and female Wistar rats. Macroscopic post-mortem examination didn’t reveal external, nor internal effects of the treatment. Haematological and biochemical blood parameters were also not impacted by the test item repeated administration.
By cons, clinical signs involved by technical impairments, lead to advise administering a minimal volume of solution for the studies implicating a repeated period of treatment.

According to these findings, the oral exposure to CuDABT during 14 days is considered as non-toxic in adult rats at 100, 300 and 1000 mg/kg/day.

Executive summary:

OBJECTIVE

The aim of this study was to evaluate the oral toxicity of a 14-day administration of Copper Diammine Bitetrazole (CuDABT) in the adult rat. This study was performed upstream of the achievement of a study following the OECD Guideline No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” adopted the 29th July 2016, to select testing doses.

MATERIALS & METHODS

Adult Wistar rats (n=3 animals/sex/experimental group) were treated by gavage with CuDABT suspensions at 0, 100, 300 and 1000 mg/kg/day daily during 14 days. Mortality and clinical signs were observed at least once a day, and body weight and food consumption recorded at least once a week. After this treatment period, animals were sacrificed for macroscopic examination, and hematological and biochemical blood analysis.

RESULTS

Analytical measurements of test item suspensions confirmed the formulations concentrations, and their homogeneity and stability in the conditions they have been used.

During the study, two females treated with 300 and 100 mg/kg/day were respectively euthanized after 10 and 12 days of treatment, because of degraded health status that occurred consecutively to defects of administration. Otherwise, no significant clinical perturbations were observed in animals, as well as no incidence on body weight and food consumption.

External and internal examination also didn’t reveal significant differences between treated and control animals.

CONCLUSION

According to these findings, the oral exposure to CuDABT during 14 days is considered as non-toxic in adult rats at 100, 300 and 1000 mg/kg/day.