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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 November to 4 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
A total of 90 Hsd: Sprague Dawley SD rats (45 males and 45 virgin females), 6 to 7 weeks old and weighing 184.2 to 195.5 g for males and 155.5 to 173.3 g for females, were received from Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy.
An acclimatisation period of 13 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations

The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C +/- 2°C and 55% +/-15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 25 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
During the pre-mating period, animals were housed 5 of one sex to a cage, in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent paper which was inspected and changed at least 3 times a week.
During the mating period, animals were housed on the basis of one male to one female in clear polycarbonate cages measuring 42x26x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
The males were re-caged after mating as they were before mating.
After mating, the females were transferred to individual clear polycarbonate solid bottomed cages measuring 42x26x18 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Suitable nesting material was provided and was changed at least 3 times a week.
Drinking water was supplied ad libitum to each cage via water bottles.
A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.

The animals arrived on 27 October 2011 and were allocated to the study on 2 November 2011. Dosing commenced on 9 November 2011 and the last necropsy was performed on 5 January 2012.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous methylcellulose
Details on exposure:
The test item was administered orally (by gavage).
Details on mating procedure:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until sperm identification and/or copulation plugs were found.
The pairing combination of any animals which have not had positive identification of mating after 14 days of pairing, was changed within each
treatment group. The subsequent pairing was monitored for mating as described above for a maximum period of 14 days.





Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The proposed formulation procedure for the test item was checked in the range from 5 mg/ml (in RTC Study no. 85950) to 100 mg/ml (in RTC Study no. 85930) by chemical analysis (concentration and homogeneity) during the pre-treatment period to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in RTC’s SOPs for concentration (90-110%) and homogeneity (CV <10%).

Samples of the formulations prepared in Week 1 and during the last week were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in RTC’s SOPs for suspensions (90-110% for concentration and CV <10% for homogeneity) with the exception of samples prepared in Week 1 that were found to be out of limits for both concentration and homogeneity. Analyses were repeated and results were found to be within the acceptability limits.
Duration of treatment / exposure:
Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy, achieving a total of 37 days of treatment.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.


Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during gestation and post partum periods up to Day 3 post partum.
During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Frequency of treatment:
once a day
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
4 groups of 10 males and 10 females received the test item by gavage at dosages of 50, 250 and 1000 mg/kg/day. A similarly constituted group of animals received the vehicle alone (0.5% aqueous methylcellulose) and acted as a control.
Control animals:
yes
Details on study design:
The oral route was selected as it is a possible route of exposure of the test item in man.
The dose levels of 50, 250 and 1000 mg/kg/day were chosen in consultation with the Sponsor based on a range-finding study (same concentrations).
Positive control:
no

Examinations

Parental animals: Observations and examinations:
Clinical signs
All clinical signs were recorded for individual animals. Once before beginning of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

Body weight
Males were weighed on the day of allocation to treatment groups, on the day that treatment beginnen, weekly thereafter and just prior to necropsy.
Females were weighed on the day of allocation to treatment groups, weekly from the first day of treatment to mating, on Days 0, 7, 14 and 20 post coitum and on Days 1 and 4 post partum.

Food consumption
Food consumption was recorded at weekly intervals whenever possible, by each cage of rats from allocation to pairing. For female animals, food consumption was also recorded on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum (starting from Day 0 post partum).
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning from the first day of treatment and up to the end of the mating period, until a positive identification of mating was made. The vaginal smear data were examined to determine the anomalies of the oestrous cycle; the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Sperm parameters (parental animals):
Parameters examined in all animals: testis weight and epididymis weight

Litter observations:
As soon as possible after parturition (Day 0 or 1 post partum), the total litter size (live and dead) was counted, sexed and examined for external abnormalities. Live pups were individually identified within the litter. All litters were weighed on Day 1 post partum. All litters were examined daily for dead and abnormal pups. The pups were also weighed on Day 4 post partum. All pups found dead were necropsied
Postmortem examinations (parental animals):
All parental animals were killed with carbon dioxide.
Parental males were killed after the mating of females (Day 37 of treatment).
Parental females with live pups were killed on Day 4 post partum. Females which had not given birth 25 days after the positive identification of mating were killed shortly after.

The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

All females killed at term (including non pregnant animals or with total resorption or total litter loss), were examined for the following:

a) external and internal abnormalities;
b) number of visible implantation sites (for pregnant animals);
c) number of corpora lutea (if detectable).

Uteri of non-pregnant females or with no visible implantations were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.
Postmortem examinations (offspring):
All viable pups were euthanised by intrascapular injection of Tanax on Day 4 post partum.
All pups found dead in the cage were necropsied. All live pups were killed on Day 4 post partum and examined for the following:

a) external abnormalities;
b) sex confirmation by gonadal inspection
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test.
Reproductive indices:
The following reproductive indices were calculated:

Males
Copulatory Index (%) = no. of animals mated x 100 / no. of animals paired

Fertility Index (%) = no. of males which induced pregnancy x 100 / no. of males paired



Females
Copulatory Index (%) = no. of animals mated x 100 / no. of animals paired

Fertility Index (%) = no. of pregnant females x 100 / no. of females paired

Males and females
Copulatory Interval = Mean number of days between pairing and mating
Offspring viability indices:
The following indices were calculated:

Pup loss at birth : (Total litter size - live litter size) x 100 / Total litter size

Cumulative pup loss on Day 4 post partum: (Total litter size at birth - live litter size at Day 4) x 100/Total litter size at birth

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Animals of both sexes did not show any sign of toxicity during the whole study. Decreased activity, dyspnea and kyphosis were the most relevant clinical signs detected in the unscheduled dead animal on Day 7 of treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One mid-dose female (no. 85950055) was sacrificed for humane reasons on Day 7 of treatment.
All females mated, although mating was not detected in one animal (no. 85950067) of the high dose group.
The number of mated females not found pregnant at necropsy was 2 in the control group (nos. 85950003 and 85950007) and one in the low dose group (no. 85950037). The number of females with live pups on Day 4 post partum was 8 in the control group, 9 in each of the low and mid-dose groups and 10 in the high dose group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain of both sexes were similar between groups.
A decrease in body weight gain detected on Day 4 post partum in the high dose group compared to controls, was considered to be of no toxicological significance. It was due to the presence of a single female (no. 85950071) with a negative body weight gain.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in food consumption in either sex.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Unscheduled deaths: The histopathological evaluation on female no. 85950055 revealed an acute inflammatory reaction in the pleura of the lungs extended in the surrounding connective tissues of the thymus and the heart. Such lesions were considered to be related to a misdosing during the administration of the test item.
Final sacrifice: No treatment-related changes were seen in selected organs/tissues (testes, epididymides and ovaries) of males or females receiving copper dibutyl dithiocarbamate, nor in the abnormalities detected in all groups at post mortem.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No treatment-related anomalies were noted in the oestrus cycle of treated females when compared to controls.
Irregular cycle (oestrus never observed) was noted in one low dose female, no. 85950025.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
The mean number of copulation plugs and mean pre-coital day’s interval were similar between groups. All females mated.
Male no. 85950022 (low dose group) did not mate after the maximum period allowed. In addition, two males in the control group (nos. 85950004 and 85950008) and one male in the low dose group (no. 85950038) with positive identification of mating did not induce pregnancy. All males induced pregnancy in mid- and high dose groups.
The number of implantations, pre-birth loss data and gestation length were similar between control and treated groups.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(parental toxicity)
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
(reproduction performance)
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical signs recorded in the pups were considered to be incidental, with no relation to the treatment of dams.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No treatment related differences on litter data and sex ratios at birth and on Day 4 post partum were detected.
An increase in pup loss from the day of birth up to Day 4 post partum was detected in the high dose group compared to controls. The increase was not statistically significant and not dose-related, thus it was considered to be of no toxicological significance. It was due to the presence of a single female (no. 85950063) with a high percentage of cumulative loss.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No relevant differences between control and treated groups were noted at necropsy in the decedent pups.
No abnormalities were recorded in the pups sacrificed on Day 4 post partum.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
On the basis of the results obtained the NOAEL (No Observed Adverse Effect Level) for males and females could be considered 1000 mg/kg/day.
 
Executive summary:

The effects of the test item, copper dibutyl dithiocarbamate, on fertility, pregnancy and early lactation of the offspring were investigated when administered orally to male and female Sprague Dawley rats.

Groups of 10 males and 10 females received the test item by gavage at dosages of 50, 250 and 1000 mg/kg/day. A similarly constituted group of animals received the vehicle alone (0.5% aqueous methylcellulose) and acted as a control. All doses were administered at a constant volume of 10 ml/kg body weight.

Males were treated for 2 weeks prior to pairing and during pairing of all females until the day before necropsy, for a total of approximately 6 weeks.

Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3post partum.

The following investigations were performed on parental animals of all groups: body weight, clinical signs, food consumption, oestrous cycle, mating performance, litter data, macroscopic observations, organ weights and histopathological examination of abnormalities. Histopathological examination of testes, epididymides and ovaries was performed only on control and high dose groups.

One mid-dose female was sacrificed for humane reasons on Day 7 of treatment. This death was considered to be related to a misdosing during the administration of the test item. Two females in the control group and one female in the low dose group were not pregnant at necropsy. Mating was not detected in one high dose female. The number of females with live pups on Day 4post partum was 8 in the control group, 9 in each of the low and mid- dose groups, 10 in the high dose group. Animals did not show any sign of toxicity. No effects on body weight and body weight gain were seen during the study. No effects on food consumption were seen during the study.

No treatment-related anomalies were noted in the oestrus cycle of treated females when compared to controls.

Pre-coital interval, copulation plugs, copulatory index and fertility index were similar between treated and control animals.

No significant differences were observed in treated and control groups for these parameters.

No treatment related differences on litter data and sex ratios at birth and on Day 4 post partum were detected.

The increase of pup loss detected from the day of birth up to Day 4 post partum in the high dose group was considered to be of no toxicological significance.

Pre-weaning clinical signs did not show treatment-related effects. Necropsy findings in decedent pups and pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.  Terminal body weight and organ weights were unaffected by treatment in both sexes. At post mortem examination, a dark colour of the spleen in 4 out of 10 males receivingcopper dibutyl dithiocarbamateat 1000 mg/kg/day was the only relevant finding detected in treated animals when compared with controls.

No treatment-related changes were seen at the histopathological evaluation in selected organs/tissues (testes, epididymides and ovaries) of males or females receiving the test item, nor in the abnormalities detected in all groups at post mortem.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

On the basis of the results obtained the NOAEL (No Observed Adverse Effect Level) for males and females could be considered 1000 mg/kg/day.