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

Endpoint summary

Description of key information

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was realised on adult Wistar rats (n=12 animals/sex/experimental group) which were daily treated orally by gavage with CuDABT suspensions at 0 and 1000 mg/kg/day up to 55 consecutive days. Mortality, body weight, clinical signs, food consumption, and sensorimotor abilities were recorded aware the experimental period. Males and females were also mated, in order to evaluate the effects of the test item on developmental parameters in the following generation.

The repeated CuDABT administration lead to observe sex-dependant difference between animals exposed to the substance and controls. In adults, body weight, food consumption, clinical signs, hematological and biochemical parameters, and coagulation time have varied. In the F1 generation, the anogenital distance and the percentage of nipple retention were also different between the two groups.

By cons, no disruption of the sensorimotor abilities and the reproductive function has been observed in adults, nor macroscopic and microscopic post-mortem abnormalities in parents and pups.

These findings have conduct to punctual discrepancies, which haven't reveal a biological disruption. No critical trouble having being demonstrated, these outcomes haven't been judged as adverse effects.

In accordance with the EC No. 1272/2008 regulation, the repeated oral exposure to CuDABT is considered as non-toxic for the reproduction and the development in rats at the dose of 1000 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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.

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

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.

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

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

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group G2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group G3

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

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.

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

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

Critical effects observed:

no

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April to July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviations have been assessed to have no significant impact on the results and the validity of the study (cf. study report attached)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Name: Copper Diammine Bitetrazole (CuDABT)
Other names/code: Copper Diamine Bitetrazole, Copper Diammoniumbitetrazole, Copper, diammine[5,5’-bi-1H-tetrazolato(2-)kN1,kN1’]
CAS No.: 538313-26-7
EC No.: 611-056-6
Batch number: P3656461
Packaging: 20 packs of 500 g
Manufacturing date: 25 August 2015
Expiration date: 25 August 2020
INERIS Identification: 16AH595 to 16AH614
An analytical certificate, not GLP-compliant, has been provided by the Sponsor

Status (20°C): blue powder
Purity: 97.4%
Water content: unknown
Molecular weight: based on the formula [Cu(NH3)2](NO3)2: 221.617 g/mol

Storage condition: Temperature and humidity restrictions have not been set. Warehouse recommendation of the test item in a depository designed for explosives in INERIS has been accepted by AUTOLIV.
Temperature was recorded by a thermometer checked and calibrated according to standard operating procedures. During the in-life study, temperatures were comprised between -4.2 and 33.3°C in the premises dedicated (Appendix 2).

Species:

rat

Strain:

Wistar

Remarks:

Wistar RjHan:WI

Sex:

male/female

Details on test animals or test system and environmental conditions:

SUPPLIER'S DATA
Supplier: JANVIER LABS
Health status : SPF (specific pathogens free)
Species/strains: Wistar RjHan:WI
Sex: males and females (nulliparous and non-gravid)
Age at the beginning of the study: 8 weeks
Number of animals: 24 males and 24 females

ACCLIMATION PERIOD
At reception, animals were weighted and placed in the acclimatization room during 6 days to allow them habituate to the laboratory ambient conditions.
Their health status was assessed by clinical observations on their day of arrival at the facility, and daily during the acclimatization period. To be identified during this phase, animals were temporary marked on the tail.

ALLOCATION IN EXPERIMENTAL GROUP
Animals included in the study were selected based on the clinical signs observed during the acclimatization period. They were distributed in the experimental groups according to the body weight recorded at the end of this period. At this time, they have been tattooed with a number on ears in order to be identified (No. 305 to 354).

ENVIRONMENTAL CONDITIONS
Animals were housed in a restricted area dedicated to rodents. The animal room conditions were set as follows:
Temperature : 20 to 24°C
Relative humidity: 40 to 70%
Light/dark cycle: 12h/12h
Ventilation: 10 to 20 cycles/hour of filtered, non-recycled air
Temperature and relative humidity were recorded continuously by a thermo-hygrograph, checked and calibrated according to an internal standard operating procedure. These parameters were daily recorded and reported in the study file. Monitoring data of artificial lighting were also reported in the facility's files.

HOUSING
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.
At the beginning of the experimental period, they were housed 2 per cage and per sex. During the mating period, males and females were housed together in the same cage (1:1). After coupling, they were placed individually, for males, to avoid risks of fighting with congeners (that may increasingly happen after mating), for females, to not disturb pregnancy and lactation.
Each cage was labeled with the study number, the experimental group number, the type of treatment and the animals' identification (number and sex).

FOOD AND WATER
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).

CONTAMINANT ANALYSES
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.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q water

Details on oral exposure:

Suspensions of the test item in Milli-Q water were prepared according to the procedure described in the previous studies on oral exposure to CuDABT.
Homogeneity and stability of the test item preparations were assessed in these studies by the measurement of total copper concentration. Based on their results, formulations between 1 and up to 200 g/L have been demonstrated to be homogenous and stable up to 7 days of storage in obscurity at room temperature.
Thus in this study, the test item suspensions were prepared once a week. Daily before dosing, they were shaken under magnetic stirring for at least 30 minutes, and continuously during administrations.
Concentrations of total copper concentrations in the formulations prepared were checked four times during the study.

EXPERIMENTAL GROUPS
The dose tested has been selected according to the results of the previous study on the oral exposure to CuDABT during 14 days. No significant difference having been observed between controls animals treated with CuDABT concerning the parameters measured (body weight, food consumption, clinical signs, mortality, macroscopic examination, biochemical and hematological parameters) whatever the three doses tested and the sex, the substance was considered as non-toxic in adult rats at 100, 300 and 1000 mg/kg/day.
Based on these results, the high-dose of1000 mg/kg/day was tested in this study. For this, two experimental groups of 12 animals/sex were tested, one treated with CuDABT at this dose level, and one control.

ADMINISTRATION
After the pre-treatment period, animals were daily dosed until the day before sacrifice. Males were treated during 35 to 37 days, dams during 49 to 55 days, and non-pregnant females during 55 days.
The dose administered corresponded to the ratio of the mass of the test item per kilogram of animals’ body weight, divided by the volume of solution per kilogram of their body weight.
During the treatment period, animals were daily dosed by gavage (using a plastic syringe fitted with a rigid stainless cannula). Each day throughout administration, the suspensions were continuously stirred to ensure an homogeneous sampling of the solution.
The volume administered has been adjusted to the most recently recorded body weight. A volume of administration of 0.5 mL/100 g of body weight was daily administered. Based on this volume, the concentration of CuDABT was adjusted in dose formulation to 200 g/Lin order to administer to animals the test item at 1000 mg/kg/day.
In the same time, controls received by gavage Milli-Q water, the vehicle used for formulation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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 (Deviation No. 10). 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:

Males were treated during 35 to 37 days, dams during 49 to 55 days, and non-pregnant females during 55 days

Frequency of treatment:

Daily

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were mated at an age they were sexually mature. Females were placed with males until the confirmation of fertilization or until 2 elapsed weeks.
Each day, a vaginal smear was performed on non-pregnant females. Gestational day (GD) 0 was defined as the day a vaginal plug was observed or as sperm was present in the smear.
The day of birth was defined as postnatal day (PND) 0.
At PND4, the size of each litter was randomly adjusted by sacrifice of the supernumerary pups. When possible, 4 pups/sex/litter were kept. No adjustment was done in litters of 8 or less pups.

Positive control:

no

Observations and examinations performed and frequency:

MORBIDITY AND MORTALITY
Each animal, adult and pup, was checked for mortality and morbidity at least once a day. The date of unscheduled death and euthanasia has been recorded, as well as animals’ body weight. A necropsy was performed when possible.

CLINICAL OBSERVATIONS
Each animal, adult and pup, 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.
Pregnancy duration was calculated from the day of fertilization to parturition.
Each litter was examined as soon as possible after birth to determine pups’ number and sex, stillbirths, little runts and the presence of abnormalities. At PND4, pups were sexed, tattooed, and their anogenital distance (AGD) measured. At PND12, the presence of nipples or areola observable in each male pup has been recorded. These manipulations were performed according to internal standard operating procedures.

VAGINAL SMEARS
Individual vaginal smears were examined daily on all females during the pre-treatment period to determine the stage of the estrous cycle, during the mating period until evidence of fertilization, and before sacrifice to determine the stage of the estrous cycle.

DETAILLED CLINICAL OBSERVATION
Detailed clinical examinations have been performed on all animals once before the beginning of the treatment period and then once a week until the end of the study. Observations and scoring were performed according to an internal standard operating procedure.

FUNCTIONAL OBSERVATIONNAL BATTERY (FOB)
Sensorimotor abilities have been assessed the weeks preceding euthanasia (two weeks before sacrifice for all males and for females No. 335, 346 and 347, and the week preceding sacrifice for the other females). A blind draw was done to select the animals to test. Ten animals/sex were selected in the control group and ten males and nine females in the group treated with CuDABT (the females have been selected on the basis they had offspring, only nine ones in this group).
Sensory reactivity has been evaluated by the animals’ response to touch, pupillary and auditory stimulations, to tail pinch and their ability to right in the mid air. The muscular strength of their fore- and hindlimbs was measured using the grip strength test. (IntelliBio, Seichamps, France). The motor activity was recorded in the open-field test (IMetronic, Pessac, France). Each animal was placed during a 60-minutes period in the dedicated arena, and their horizontal (total distance covered and distance covered in the peripheral area) and vertical (total number of rearing and number of rearing in the peripheral area) locomotion automatically recorded by the device.
These evaluations have been performed according to internal standard operating procedures.

PHYSIOLOGICAL PARAMETERS
The rectal temperature of the animals selected for sensorimotor investigations was measured immediately after the tests precited with a dedicated device (rectal probe for rats RET-2 Type T thermocouple, and thermometer 8851 K.J.T., BIOSEB, Vitrolles, France), according to an internal standard operating procedure.

BODY WEIGHT
The body weight of each adult animal was recorded the day of arrival, the day of randomization, before the beginning of the treatment period, at least once a week until the end of the study, and before euthanasia.
Pregnant females were additionally weighted at GD0, 7, 14 and 20, within 24 hours after parturition (PND0 or 1), and at PND4 and 13.However, the body weight of certain females was recorded at GD1 or 2, instead of GD0, or not recorded before GD7 (Deviation No. 2).
Each litter was weighted as soon as possible after birth. Pups were individually weighted at PND4 and 13.

FOOD CONSUMPTION
Food consumption has been recorded once a week along the study, except during the mating period.

BLOOD COLLECTION AND ANALYSIS
Prior to sacrifice, animals were fasted overnight for at least 14 hours.
After euthanasia, their blood has been collected at the vena cava into tubes containing an anticoagulant (EDTA tubes for hematology, lithium heparin tubes for biochemistry and sodium citrate tubes for the analysis of coagulation).
Analysis was performed the day of blood collection, except for thyroid hormone level of pups at PND13, which was quantified later. In the wait, plasma samples were stored at -20°C.
The results of five adult animals/sex/experimental group, randomly selected in the batch of animals having performed FOB’s tests, have been statistically analyzed for hematological, coagulation and biochemical parameters.

HEMATOLOGY
The following parameters have been measured using a hematology analyzer (MS9-5 Vet, MSTM Laboratories, Osny, France), and according to an internal standard operating procedure: hematocrit, hemoglobin concentrations, erythrocytes and reticulocytes count, total and differential leucocytes count, and platelet count.

COAGULATION
The prothrombin time has been determined using a coagulometer (KHB202-4, Kinghawk, Jinan, China) and a commercial reagent kit according to an internal standard operating procedure.

BIOCHEMISTRY
A serum chemistry analyzer (M-SCAN II, MSTM Laboratories, Osny, France) and commercial kits have been used to measure the following parameters according to an internal standard operating procedure: sodium, potassium, chloride, total CO2, Na/K (IONO crown), 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 (VET16 crown).

THYROID HORMONES
The thyroid hormone level has been measured, using the chemistry analyzer and a commercial kit on five adult animals/sex/experimental group (the same animals as previously selected), as well as on pooled samples of 2 pups per litter at PND4 (collected only in litters constituted of more than 8 animals), and on 2 pups/litter (1 male and 1 female when possible) at PND13.

Sacrifice and pathology:

NECROPSY PROCEDURE
On completion of the study, all adult animals and pups at PND13 have been euthanatized by an intraperitoneal injection of sodium pentobarbital (100 mg/kg of body weight).
Pups sacrificed at PND4 were anesthetized by isoflurane inhalation (induction at 4% with an air flow of 2L/min) and euthanized by decapitation, as validated by the regional ethics committee.

MACROSCOPIC POST-MORTEM EXAMINATION
A complete macroscopic post-mortem examination has been performed on all animals, except the sciatic nerve, skeletal muscle and femoral bone with bone narrow, which were observed only in five adult animals/sex/group.
This exam 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, as well as organs and tissues listed in the tissue procedure table below.
Special attention has been paid to the organs of the reproductive system. The number of implantation sites was also recorded in all females of the parental generation.
Pups dead during the study and euthanized at PND4 and 13 have also been examined for potential external gross abnormalities.
During dissection and collection, certain organs and tissues have been blessed or were lost.

ORGAN WEIGHTS
Organs and tissues listed in the tissue procedure table have been weighted immediately after dissection, except thyroid glands which were measured after fixation to avoid tissue damage. The ratio of organ weight to the body weight recorded before sacrifice has then be calculated.

PRESERVATION OF TISSUES AND SLIDE PREPARATION
Organs and tissues specified in the tissue procedure table have been fixed in 4% buffered formalin, except testes and kidneys which were preserved in Bouin’s fixative.
Femoral bones were decalcified after fixation.
All specimens were then dehydrated and embedded in paraffin wax blocks according to an internal standard operating procedure.
Organs and tissues sectioning and slides staining were performed according to their standard operating procedures.

MICROSCOPIC EXAMINATION
A microscopic examination was performed on gonads and accessory sex organs from all adult animals, on thyroid glands from all adults, and from one pup/sex/litter at PND13, and on all organs and tissues listed in the tissue procedure table from five adult animals/sex randomly selected in each experimental group.
The histopathological assessment was performed according to his standard operating procedures.

Statistics:

Data were manually recorded in study books prepared before the beginning of the experiment and then copied into Excel files to be analyzed.
Effects of the test item were analyzed for each sex, when applicable, by mean of a Student t-test for quantitative variables and using the Pearson's Chi-square test for qualitative data; with the control group used as reference. 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

ADULT ANIMALS
A specific sign was noted in a majority of rats treated with CuDABT that was not recorded in controls. During several days along the study, the presence of feces of abnormal color was observed in the home cage of these animals, especially in males. The blue color of the test item powder leads to suppose that this was caused by the excretion of the substance. This observation having been transient, animals have seemed to habituate to the treatment.
Otherwise, some clinical signs related to respiratory disturbance (red stains on nose, noisy respiration, nasal secretion) have been observed throughout the study. These manifestations having been observed in rats of the two groups, they seemed to be consequent to gavage defects.
In addition, the female No. 347, from the group treated with CuDABT, presented several signs of health degradation (respiratory distress, mouth secretion, hunched posture, and thin body condition). Because limit endpoints have been reached, the treatment was stopped during several days to limit suffering. Her condition having ameliorated, this female was treated again and kept in the study. A defect in the gavage administration was probably also the cause of her health degradation.
Alopecia was also observed in female control animals. This phenomenon is, first in females, a marker of dominance regarding congeners, and, secondly, especially observed during the perinatal period, since it’s a trait of maternal behavior.
Other observations, recorded only once during the study (blood on fur, wound, and eyelid partially closed), were not considered to be significant signs of trouble.

PUPS
Few abnormal clinical signs were recorded in pups during the study.
At birth, one control female, found dead, presented a flattened head. Two little runt females were also recorded in this group. One female treated with CuDABT was crumpled and blue, and one male cold at PND0.
The observation of some abnormalities in pups at birth being normal, the incidence in this study being very low, no effect of the treatment can be suspected to having induced these anomalies.

TREATMENT ADMINISTRATION
Difficulties of gavage were encountered aware the study. Defects of administration induced aggressiveness in animals, whose struggled during treatment. Degraded health status having also being observed in certain animals, they were not dosed some days to limit suffering. Regurgitations were also observed all along the experimental period in the two groups.

DETAILLED CLINICAL OBSERVATIONS
The principal and most frequent signs observed were:
-hyper- and hypoactivity (observations especially made when the animals were placed in the standard area), and hypervigilance,
-reaction to cage stimulation, to cage withdrawal, and to handling (vocalizations, twitching, twisting, noisy breathing, hyper reactivity, escape, difficult to seize, and muscle hypertonia),
-alopecia (localized on fur, legs, face, and abdominal area),
-noisy breathing and sneezing,
-excessive and abnormal excretion (feces of abnormal color and aspect, urination in the standard area).

Other punctual observations were also recorded:
-trouble of locomotor coordination (crawling),
-wound,
-red stains in nasal cavity,
-teeth gnashing,
-abnormal posture (huddled up and hunched animals),
-occurrence of involuntary movements (nodding),
-isolation from the congener in the home cage,
-maternal behavior impairment (one pup isolated from the nest),
-under-conditioned corpulence,
-enophtalmia.

As described previously, an excessive excretion of feces was observed in the evaluation of detailed clinical signs in animals treated with CuDABT compared to controls. Indeed, a large amount feces of abnormal aspect and color was reported when animals were placed in the standard area.
In both group, the hyper reactivity to manipulation (cage stimulation and withdrawal, and handling) and the changes in the level of activity observed (hyper- and hypoactivity, and hypervigilance) were probably the consequences of an increase of their stress level, that was due to their daily important stimulations.
Moreover, the behavior of all animals may have been influenced by the housing conditions, which have varied aware the study (animals initially housed at two per cage per sex, then per couple, and finally individually).
Except an abnormal fecal excretion, which was as consequence of the treatment, the clinical signs recorded have been globally observed in animals of the two groups and the two sexes.

Mortality:

no mortality observed

Description (incidence):

No adult animal died, nor has been euthanized before the end of the study because of limit endpoints.
Four pups (one male and two females from the control group, and one male from the group treated with CuDABT) were found dead at birth, and five were missing between PND0 and 4. Dams have probably murdered them because of a poor health.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

1. Adults
No difference was observed in the body weight of males of the two groups all along the study period.
By cons, the statistical analysis revealed a significant difference in females at the time of sacrifice. The females treated with the test item showed a lower body weight compared to controls. No significant variation has been observed the weeks before, so this result cannot be consistently interpreted as a consequence of the test item treatment.

2. Females during perinatal period
Body weights recorded at GD0, 1 and 2 were not analysed statistically, because of potential bias in the comparison of data.
At GD20, a significant difference was observed between the two groups. Females treated with CuDABT showed a lower body weight compared to controls. Body weight of females being very variable the days before parturition, because it depends on the number of pups, and of their proper body weight, this difference cannot be strictly related to the treatment.

3. Litters and pups
The body weight of litters (sum of the body weight of all pups) at PND0 and 4 hasn’t been significantly different between controls and animals treated with CuDABT.
By cons, pups individual body weight differed significantly between the females of the two groups at PND4, whereas no variation was observed in male pups. At PND13, no difference was noted between the two groups, whatever the sex. This difference having being transient, since no more difference were recorded at PND13, no significant impact of the test item can be conclude.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean quantity of food consumed per day was significantly higher in males treated with CuDABT compared to controls during four weeks aware the study (the two weeks preceding mating, and the two weeks before sacrifice). By cons, no significant difference was observed between the females of the two groups during the entire experimental period.
This increase of food consumed, not related to a variation of body weight, was potentially indirectly induced by an increase in the metabolism of the substance (as already discussed to interpret the increase of excretion recorded in the observation of the clinical signs), and the need for animals to fill the lack of excreted nutrients.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Concerning hematological parameters, only one level, related to the erythrocytes concentration in blood samples, was significantly higher in males treated with CuDABT compared to controls.
Regarding coagulation, the prothrombin time was significantly lower in males treated with the test item rather than controls, whereas no difference was observed in females.
No variation having being observed at the clinical level, nor after the macro- and microscopic examination of organs and tissues, between animals of the two groups, no significant adverse trouble can be discussed.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

1. Biochemical results
In males treated with the test item, only one parameter, the total bilirubin concentration in plasma, was significantly lower in comparison to controls. No variation having being observed elsewhere, it isn't relative to a significant biological disruption.
By cons in females, the concentrations of urea, calcium, albumin and the ratio urea/creatinine were significantly different between animals of the two groups. Indeed, all these parameters were lower in females treated with CuDABT compared to controls.
The bibliography reports that hypocalcemia is commonly observed during the perinatal period, and especially at the onset of lactation (Weaver et al. 2016, Yamagishi et al. 2007). Indeed, pregnant and lactating females are particularly vulnerable, because of important metabolic changes occurring during this period, mobilizing maternal energy and calcium reserves.
No clinical signs (like muscular trouble), nor macro- and microscopic abnormalities (affecting kidneys, bones, thyroid glands, and gastrointestinal tract, as commented in the § 12.4.2. and 12.4.3.), which could be disrupted in relation to hypocalcemia, have been observed. No teratogene effects in pups of the F1 generation have also been highlighted. Moreover, additional measures on the non-pregnant females of the study revealed the same biochemical levels as controls. Results of the 14-day oral exposure to CuDABT haven’t also shown a variation in calcium concentration, whatever the dose and the sex.
So, this dysregulation, which is a usual perinatal deficit, has probably been exacerbated by the high dose intake of CuDABT, but regarding the absence of significant adverse consequences, it was not considered as a real toxic effect of the substance.

2. T4 levels
The level of T4 thyroid hormone in plasma hasn’t been significantly different between controls and CuDABT-treated animals, whatever the sex. No variation has been observed in adult animals, nor in pups at PND4 and 13.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The scoring used to evaluate sensory reactivity tests, the individual data of sensorimotor activity, a summary of the records, and means of muscular strength and motor activity are presented in teh final report.
The statistical analysis of reactivity to visual, auditory and sensory stimuli (evaluate in response to touch, pupillary and auditory stimulations, to tail pinch and righting reflex), muscular strength of fore- and hindlimbs, and motor activity (assessed in the open-field), hasn’t showed significant difference for all the variables recorded between control and animals treated with CuDABT, whatever the sex.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No difference between controls and animals treated with CuDABT was observed concerning the weight of organs, nor on the relative organs weight, whatever the sex.
Thyroid glands weight and relative weight haven’t also differed in pups of the two groups at PND13.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

1. Adults
The external exam in adult animals provided the following results:
-alopecia,
-noisy respiration,
-enophtalmia.

The internal examination revealed the following abnormalities:
-liver of abnormal colour, one testis, one epididymis and the prostate of abnormal size,
-lack of right kidney and right horn,
-dark spots on lung,
-uterus horns of abnormal size,
-ovaries of abnormal size.

These abnormalities having being observed both in controls and animals treated with the test item, it supposes they were not related to the treatment administered, but rather on biological spontaneous occurrences.
Thus, no macroscopic significant difference between the two groups was revealed by the external and internal examinations in adult animals (Tables 47 and 48).

2. Pups
The macroscopic observation of pups hasn’t also revealed significant difference between controls and animals treated with the test item.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Cf. behaviour.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Some technical problems were encountered at the sectioning stage. Nevertheless, histopathological microscopic examination has been performed and consistent results have been obtained.
Two study phase reports, one per the test site, summarized the histopathological analysis in terms of materials and methods, observations, results, and conclusions.
The histopathological exam revealed no significant histological defects in the organs of adult animals exposed to CuDABT, nor on the thyroid glands of their offsprings.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

REPRODUCTION AND DEVELOPMENTAL PARAMTERS
Individual parameters related to reproduction and development in adults of the parental generation and in pups, a summary report of the test item effects on reproduction and development.

1. Reproduction parameters

Concerning fertility, no significant variation of the duration of the oestrus cycle, nor of its regularity, has been observed between the two groups. Only two females presented an irregular cycle. Despite this trouble, the female from the control group had offspring.
Fertilization was observed after 1 to 3 days of mating (conceiving days), except for one femal, in who it was observed after 15 days. Eleven females gave birth in the control group and nine in the group treated with CuDABT. Pregnancy duration was between 21 and 22 days. No significant difference between animals of the two groups was observed concerning these parameters of reproduction.
The number of implant sites observed at necropsy, as well as of live pups at PND0 and 4, were not significantly different between controls and females treated with the test item.
No difference between the two groups was also observed in the number of live pups at PND0 and 4, of animals found dead at birth, of abnormalities, and of pre- and postnatal offspring loss.
The number of males and females, and the sex ratio calculated at PND0 and 4 were as well not significantly different between controls and animals treated with CuDABT.

2. Developmental parameters
Concerning the measure of the AGD, females treated with CuDABT presented a distance between the middle of the anus to the genital tubercle significantly longer than controls.
For nipple retention, the percentage of nipple and/or areola observed in males was statistically higher in the pups of the group treated with the test item, compared to controls. Indeed, they persisted in 100% of the animals from the CuDABT-treated group, against 68.2% in the control group.
AGD and nipple retention are dedicated to the detection of potential endocrine disruption substances (Christiansen et al. 2014, Gray et al. 2000).
Concerning the AGD, a narrowing of the distance in males could have been expected as a toxic effect. The difference highlighted in females may be an artefact due to the technical difficulty of measuring this distance.
No significant difference in the AGD having being observed between controls and male pups exposed to CuDABT, the observation of nipple retention seemed likely reflect a delay in their development, rather than a feminization, and so a toxic effect of the substance.
Delays in the maturation of offspring in their first days of life (ie. before weaning) are very common and usually transient, because dependent on factors as the litter and the body weight (where a slight difference can punctually lead to observe a developmental gap between pups, without being a significant biological trouble).

Details on results:

Eight suspensions of the test item were prepared to ensure the treatment of the adult animals all along the study.
The analysis of four aliquots of these preparations showed a coefficient of variation in the range of acceptability of 15% (validity interval: 200 ± 30 g/L). These results validated the test item concentration at the desired dose level in the formulations administered to animals.
According to the findings of the acute oral study on CuDABT, the formulation of 200 g/L in Milli-Q water having being considered homogeneous and stable in time, a fresh solution was prepared once a week to cover 7 days of animals' dosing.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

dermal irritation

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

water consumption and compound intake

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Conclusions:

The oral daily repeated treatment to CuDABT at 1000 mg/kd/day in the Wistar rat significant sex-dependant difference between animals exposed to the substance and controls. In adults, body weight, food consumption, clinical signs, hematological and biochemical parameters, and coagulation time have varied. In the F1 generation, the anogenital distance and the percentage of nipple retention were also different between the two groups.
By cons, no disruption of the sensorimotor abilities and the reproductive function has been observed in adults, nor macroscopic and microscopic post-mortem abnormalities in parents and pups.
These findings have conduct to punctual discrepancies, which haven't reveal a biological disruption. No critical trouble having being demonstrated, these outcomes haven't been judged as adverse effects.
In accordance with the EC No. 1272/2008 regulation, the repeated oral exposure to CuDABT is considered as non-toxic for the reproduction and the development in rats at the dose of 1000 mg/kg/day.

Executive summary:

OBJECTIVE

The aim of this study was to evaluate the repeated oral toxicity of Copper Diammine Bitetrazole (CuDABT) on reproduction and development in rat. This study was performed in accordance to the OECD Test Guideline No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” adopted the 29 July 2016.

MATERIALS & METHODS

Adult Wistar rats (n=12 animals/sex/experimental group) were daily treated orally by gavage with CuDABT suspensions at 0 and 1000 mg/kg/day up to 55 consecutive days. Mortality, body weight, clinical signs, food consumption, and sensorimotor abilities were recorded aware the experimental period. Males and females were also mated, in order to evaluate the effects of the test item on developmental parameters in the following generation. At sacrifice, analysis of hematological, biochemical, in particular the T4 thyroid hormone level, and coagulation parameters were performed on blood. Post-mortem exams were also performed to highlight potential macroscopic and microscopic changes on organs and tissues, regarding especially the reproductive system in adults and thyroid glands in pups.

RESULTS

The repeated CuDABT administration lead to observe significant sex-dependant difference between animals exposed to the substance and controls. In adults, body weight, food consumption, clinical signs, hematological and biochemical parameters, and coagulation time have varied. In the F1 generation, the anogenital distance and the percentage of nipple retention were also different between the two groups.

By cons, no disruption of the sensorimotor abilities and the reproductive function has been observed in adults, nor macroscopic and microscopic post-mortem abnormalities in parents and pups.

CONCLUSION

These findings have conduct to punctual discrepancies, which haven't reveal a biological disruption. No critical trouble having being demonstrated, these outcomes haven't been judged as adverse effects.

In accordance with the EC No. 1272/2008 regulation, the repeated oral exposure to CuDABT is considered as non-toxic for the reproduction and the development in rats at the dose of 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

System:

other: No adverse effected observed among all parameters followed in the Combined Repeated Dose Toxicity Study

Justification for classification or non-classification

In accordance with the EC No. 1272/2008 regulation, the repeated oral exposure to CuDABT is considered as non-toxic for the reproduction and the development in rats at the dose of 1000 mg/kg/day.