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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Élevage CHARLES RIVERS, Domaine des Oncins BP 109, 69592 L’Arbresle Cedex, FRANCE
- Age at study initiation: 8 wks
- Fasting period before study: no fasting period
- Housing: Rats were housed in a restricted area reserved to rodents and they were housed in polycarbonate cages (Makrolon: type 3) containing autoclaved dust-free bedding (sawdust + chips). Cages were changed at least once a week.
- Diet (e.g. ad libitum): Specific rat food (Genestil) was dispensed ad libitum to the animals during the study in stainless troughs
- Water (e.g. ad libitum): All animals had free access to filtered tap water, contained in polycarbonate bottles supplied with stainless teats
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 40% to 70%
- Air changes (per hr): 15 to 20 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h / 12h

IN-LIFE DATES: From: 2013-02-05 To: 2013-04-12

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a homogeneous suspension in drinking water treated by reverse osmosis. The test item was mixed under magnetic stirrer (for homogeneous suspensions) with the required quantity of vehicle. The frequency of dose formulation preparation (1 preparation for 7 days) was based on the results of the stability assay realized by CiToxLAB France (Study No 39705 AHS), stating that dose formulations containing CuGUN ranging from 2 mg/mL to 200 mg/mL are considered to be suitable for routine administration in GLP toxicological studies, for up 7 days after preparation.
Concentration of the test item was assessed in drinking water treated by reverse osmosis. This information was required by GLP to ensure the quality of the test item preparation administered to rat.

VEHICLE
- Concentration in vehicle: 3, 10 and 30 g/L of test item
- Amount of vehicle (if gavage): 10 ml/kg/day

ADMINISTRATION
The dose formulations were administered by esophageal intubation using a rigid cannula. The exact volume to administrate to each animal was calculated based on the most recently recorded body weight.
The dose formulations were stirred continuously throughout the dosing procedure and were allowed to attain room temperature prior to their administration to animals.
Control animals were received the vehicle only.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Females were placed with the same male until pregnancy occured or until 2 weeks have elapsed
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): 1 female/cage
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each dose tested, dose preparation aliquots were taken from the middle of the flask, on the 1st, 3rd and 7th preparation. CuGUN concentration was determined by the chemical analysis laboratory in INERIS, according to the method described and validated in the rapport study DRC-12-120813-11162A “Validation of the dissolved copper analytical method"

Duration of treatment / exposure:

Males were dosed for 2 weeks prior to mating, during the mating period, and until the end of the study.
Females were dosed throughout the study. This included 2 weeks prior to mating, the variable time of conception (up to 14 days mating), the duration of pregnancy and 4 days after delivery, up to and including the day before scheduled death.

Frequency of treatment:

Daily, on the morning, approximately at the same time

Details on study schedule:

All animals were dosed after the acclimation period, for 14 days prior to mating. Day 1 corresponds to the first day of the treatment period.
Mating began after the animals have attained full sexual maturity (10 weeks for Wistar male and female rats Crl:WI(Han)).
Dosing were continued in both sexes during the mating period.
Females were placed with the same males until pregnancy was occured or until 2 weeks have elapsed.
Each morning, the females were examined for the presence of sperm. Day 0 of pregnancy was defined as the day sperm was found.
Female showing no-evidence of copulation and males were dosed after the mating period until the end of the study and were euthanized.
Daily dosing of pregnant females were continued throughout pregnancy and until Day 3 post-partum. The day of birth (when parturition is complete) were defined as Day 0 post-partum. Dams with offspring were euthanized on Day 4 post-partum.

Remarks:

Doses / Concentrations:
30 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested
gavage

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested
gavage

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results from the OECD Guideline No 407 “4-week toxicity study by the oral route (gavage) in rats of CuGUN” realized by CiToxLAB France (study No 39682 TSR). The control group was received the vehicle only (drinking water treated by reverse osmosis).
- Rationale for animal assignment (if not random): The Study Director selected the study animals on the basis of clinical observations done during the acclimation period. The animals were distributed into study groups, based on the body weights assessed the day of selection. All individual body weights were within ± 20% of the average body weight.

Positive control:

no positive control

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: on the day of arrival, on the day of selection, from Day 1 to termination: at least once a day, and more frequently when signs of toxicity are observed.
- Cage side observations: Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality, were
recorded. These records included time of onset, degree and duration of toxicity signs.


DETAILED CLINICAL OBSERVATIONS: No
- Time schedule: The duration of gestation was recorded and was calculated from Day 0 of pregnancy. Each litter was examined as soon as possible after delivery to
establish the number and sex of pups, still births, live births, runts (pups that are significantly smaller than corresponding control group) and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighted within 24 hours after parturition (Day 0 or Day 1 post-partum) and on Day 4 post-partum. In
addition to the observations on parent animals, any abnormal behavior was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of arrival, on the day of selection, on Day 1, before the first treatment, weekly and at termination. During pregnancy, females will be weighted on Days 0, 7, 14 and 20 and within 24 hours of parturition (Day 0 or Day 1 post-partum) and Day 4 post-partum

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/week: Yes
- Time schedule: weekly, except during the mating period

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
right and left testis weight, right and left epididymis weight were recorded

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
complete litters resorptions, complete litters cannibalized, dams with live young born, dams with live young born at day 4 post partum, livepups per dam at birth (mean), live pups per dam at 4 day post partum (mean), sex ratio (m/f) at birth (mean), sex ratio (m/f) at 4 day post partum, litter weight at birth (mean), litter weight at 4 day post partum (mean), pup weight at birth (mean), pup weight at 4 day post partum (mean), dams with abnormal pups

GROSS EXAMINATION OF DEAD PUPS: no

Postmortem examinations (parental animals):

SACRIFICE
Special attention were paid to the organs of the reproductive system. The number of implantation sites and the number of corpora lutea will be recorded in parental females.
Ovaries, testes, epididymis, accessory sex organs and all organs showing macroscopic lesions were collected, preserved in buffered 10% formalin or in Bouin’s fixative (testes, epididymis) and prepared for histo-pathological analysis.

GROSS NECROPSY
- Adult animals will be examined macroscopically for any abnormalities or pathological changes. This were included examination of the external
surfaces, all orifices, the cranial cavity and the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Appendix 6 of the final report were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- Dead pups and pups euthanized on Day 4 post-partum will be carefully examined externally for gross abnormalities.

Statistics:

Data were manually recorded in study books prepared before the study. These data were then transferred into computer files (Excel format) and analyzed using either Excel and/or GraphPad Prism® softwares.

Reproductive indices:

Pairs started (n)
Vaginal sperm detected (n)
Female achieving pregnancy
Conceiving days 1-5 (n)
Conceiving days 6 and up (n)
Pregnancy = 21 days (n)
Pregnancy = 22 days (n)
Pregnancy = 23 days (n)
Complete litter resorptions
Complete litters cannibalized
Dams with live young born (n)
Dams with live young at day 4 post partum
Corpora lutea per dam (mean)
Implants per dam (mean)

Offspring viability indices:

Complete litter resorptions
Complete litters cannibalized
Dams with live young born (n)
Dams with live young at day 4 post partum
Corpora lutea per dam (mean)
Implants per dam (mean)
Live pups per dam at birth (mean)
Live pups per dam at day 4 post partum
Sex ratio (m/f) at birth (mean)
Sex ration (m/f) at day 4 post partum (mean)
Litter weight at birth (mean)
Litter weight at day 4 post partum (mean)
Pup weight at birth (mean, g)
Pup weight at day 4 post partum (mean, g)
Dams with abnormal pups
Preimplantation loss (corpora lutea minus implantations)
Postimplantions loss (implantations minus live births)
Post-natal loss (live birth minus alive at day 4 post partum)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Some effects were observed:
- group 1 (control):
- group 2 (300 mg/kg) : thin hair coat (3/10), loss waight (1/10), noisy respiration (2/10), brown mouth outlines (1/10), abnormal respiration (1/10), brown stains on nose (1/10)
- group 3 (100 mg/kg) : thin hair coat (1/10), piloerection (1/10) decrease activity (1/10)
These effects were considered not relevant for NOAEL determination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male rats BW decreased for the 300 mg/kg group whereas no effect was observed for female.
These effects were considered not relevant for NOAEL determination.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

FC decreased for the male rats treated at 300 mg/kf of CuGUN during the first week of exposure (no effect on the 2nd, 3rd and 4th week of exposure) and for the female at 300 mg/kg during the first and third week of exposure (no effect on the 2nd and 4th week of exposure).
These effects were considered not relevant for NOAEL determination.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No significantly effects

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No significantly effects

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No significantly effects

Reproductive performance:

no effects observed

Description (incidence and severity):

No significantly effects

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No effect

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): BW gain of male rats was significantly reduced in the high-dose group (300 mg/kg), food consumption in males was significantly decreased from controls in the high-dose group (300 mg/kg) in the first dosing week (0-7 day). In females, the high-dose group showed significantly decreased food consumption in the second ans fourth dosing week.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

ORGAN WEIGHTS (PARENTAL ANIMALS)

GROSS PATHOLOGY (PARENTAL ANIMALS)

HISTOPATHOLOGY (PARENTAL ANIMALS)

OTHER FINDINGS (PARENTAL ANIMALS)

Key result

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

VIABILITY (OFFSPRING)

CLINICAL SIGNS (OFFSPRING)

BODY WEIGHT (OFFSPRING)

SEXUAL MATURATION (OFFSPRING)

ORGAN WEIGHTS (OFFSPRING)

GROSS PATHOLOGY (OFFSPRING)

HISTOPATHOLOGY (OFFSPRING)

OTHER FINDINGS (OFFSPRING)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

Reproductive effects observed:

not specified

Conclusions:

Under the experimental conditions of this study, the test item, CuGUN at a dose og 300, 100 and 30 mg/kg did not cause reproductive and developmental toxicity by oral exposure. Therefore, according ti the regulation 1272/2008/EEC, the classification of the test item is "not classified" based on the reproduction/developmental toxocity screening test (OECD guideline n°421)

Executive summary:

The objective of this study was to evaluate the reproduction and developmental toxicity of the test item Copper Guanylurea Nitrate (CuGUN), in Wistar rat. The study design was based on OECD Guideline No. 421.

This reproduction/developmental toxicity screening test is designed to generate information concerning the effects of CuGUN on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Method

Three doses of CuGUN were administered (300, 100 and 30 mg/kg/day bodyweight) to groups of males and females.

Males were dosed for 2 weeks prior to mating, during the mating period, and until the end of the study.

Females were dosed throughout the study. This included 2 weeks prior to mating (with the objective of covering at least two complete oestrus cycles), the variable time of conception (up to 14 days mating), the duration of pregnancy and 4 days after delivery, up to and including the day before scheduled death.

Results

Male and female WISTAR rats were treated with 300, 100 and 30 mg/kg/day of CUGUN

No unscheduled deaths, no clinical signs, no macroscopic changes and no microscopic changes considered to be related to the treatment of CuGUN were observed on parent animals and their offspring treated, during the observation period.

Conclusion

Under the experimental conditions of this study, the test item, CuGUN at a dose of 300, 100 and 30 mg/kg/day did not cause reproductive and developmental toxicity by oral exposure. Therefore, according to the regulation 1272/2008/EEC, the classification of the test item is "not classified" based on the reproduction/developmental toxicity screening test (OECD guideline N°421)

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

Only one study available but quality is sufficient for no observed effect level determination.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There were no adverse effects observed in the repeated dose toxicity study in reproductive organs of tissues or any adverse effects in the screening study for reproductive toxicity.

No data are available for other routes of exposure (inhalation and dermal route). However, as no reproductive effect was observed for oral exposure, no reproductive effect is expected by inhalation and dermal route. Indeed, the exposure by inhalation is expected to be very 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). According to the acute toxicity study results, CuGUN seem to be more toxic by oral route than dermal route. This is probably due to a more important absorption by oral route than dermal.

Short description of key information:
No unscheduled deaths, no clinical signs, no macroscopic changes and no microscopic changes considered to be related to the treatment (30, 100 and 300 mg/kg/day) were observed on parent animals and their offspring treated during the observation period. Therefore, under the experimental conditions of this reproduction/developmental toxicity screening test, the test item, CuGUN at a dose of 300, 100 and 30 mg/kg/day did not cause reproductive toxicity by oral exposure.

Justification for selection of Effect on fertility via oral route:
The study was performed in accordance to OECD 421 and to GLP. No deviation was observed.

Effects on developmental toxicity

Description of key information

No unscheduled deaths, no clinical signs, no macroscopic changes and no microscopic changes considered to be related to the treatment (30, 100 and 300 mg/kg/day) were observed on parent animals and their offspring treated during the observation period. Therefore, under the experimental conditions of this reproduction/developmental toxicity screening test, the test item, CuGUN at a dose of 300, 100 and 30 mg/kg/day did not cause developmental toxicity by oral exposure.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There were no adverse effects observed in the screening study for developmental toxicity.

No data are available for other routes of exposure (inhalation and dermal route). However, as no developmental effect was observed following oral exposure, no developmental effect is expected by inhalation and dermal route. Indeed, the exposure by inhalation is expected to be very 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). 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 classification or non-classification

Under the experimental conditions of this study, the test item, CuGUN at a dose of 300, 100 and 30 mg/kg/day did not cause reproductive and developmental toxicity by oral exposure. Therefore, according to the regulation 1272/2008 (CLP), the classification of the test item is "not classified" based on the reproduction/developmental toxicity screening test (OECD guideline N°421).