Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Two screening tests are available to evaluate the reprotoxicity of copper dibutyldithiocarbamate. No adverse effect were observed on both studies on parental toxicity and on reproduction parameters. Based on these results, the NOAEL for the maternal toxicity and the NOAEL for the reproduction performance is 1000 mg/kg/d in rats.

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
yes
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.
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
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
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
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: 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.
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
Critical effects observed:
no
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
Critical effects observed:
no
Reproductive effects observed:
no
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.

 

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1997
Deviations:
yes
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan Charles River Laboratories International, Inc. (Atsugi Production Facility)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old
- Weight at study initiation: Males: 319~368g, Females: 212~249 g

- Housing: Male and female, 1 each, were housed per cage in the mating period. 1 litter was housed during the lactation period, and 1 animal was housed in the other periods, including the quarantine period.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum ; Polycarbonate water bottles (200 and 700 mL) were used, and replaced on the date of grouping and subsequently at a frequency of 1 time within 7 days from the administration start date.
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0~25.0°C
- Humidity (%): 35.0~75.0
- Air changes (per hr): 6~20 times/hour, all-fresh air supply
- Photoperiod (hrs dark / hrs light): 12 hours/day (7:00~19:00)
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.1% w/v Tween 80-spiked 0.5% w/v CMC-Na aqueous solution (Tween 80: Kanto Kagaku K.K., Lot No.: 807X1315, CMC-Na: Wako Pure Chemical Industries, Ltd., Lot No.: SDL2940)
Details on exposure:
Administration was made using a disposable syringe with a feeding tube attached. The administration solution was used while thoroughly agitating with a stirrer.
Details on mating procedure:
Male/female 1:1 mating pairs were set with the males and the female test animals of each group, and cohabited for a maximum of 7 days and nights from the evening of day 15 (mating start date). From the day after the start of mating, vaginal smears were collected during the AM hours for the females, and mating was established if a vaginal plug or sperm in the vaginal smear sample were seen, and that day was treated as Day 0 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (n=3) taken from three points in the Administration Solution at initial preparation (top layer, middle layer, and bottom layer) were analysed according to the methods of Item 7.7. As a result, the average concentration (actual measurement: 91.0~93.8%, within ±10% of set concentration) and uniformity (actual measurements: 0.2%~0.9%, within 10% C.V. of the analysis values of the top layer, middle layer, and bottom layer), were confirmed as being within the allowable range (Item 12.4).
Duration of treatment / exposure:
Males: A total of 42 days from 14 days before mating, through the mating period, and up to the day before autopsy.
Females : This was the 14 days before mating, through the mating period, Gestation period, and parturitions, up to the 4th day of lactation (treating the date of parturition as lactation day 0). However, this was up to 25 days after confirmation of mating for non-pregnant animals.
Satellite animals (females): Up to the 42nd day without mating.
Frequency of treatment:
daily
Details on study schedule:
All mated females were allowed to deliver naturally. The observation of the parturition was performed twice per day (9AM and 4PM) from day 21 of Gestation until day 25. Those animals that had completed parturition at the 9AM point were treated as pregnant at that date, and that date was treated as lactation day 0. Furthermore, animals where parturition was completed at the time of 4PM were observed with the following day as lactation day 0. If there is no parturition even after 25 days have passed after mating confirmation were treated as non-pregnant females. The pregnant animals (dams) were allowed to nurse neonates up to 4 days postpartum (lactation day 4), and observation made ach day for the presence of lactation statuses such as lactation, nesting, and cannibalism, etc.
Dose / conc.:
40 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
400 mg/kg bw/day
No. of animals per sex per dose:
females : 12 / dose
males : 7/ dose (control and high dose groups), 12/dose (low and medium dose)
satelite groups (control and high dose groups): 5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
A 14-day recovery period after the end of the Dosing Period was set for 5 males and females, respectively, of the control group and 1000 mg/kg group. However, the females were set as a satellite where mating was not performed.
The following items were examined. The recording of the dates for the males and female satellite animals treated the administration start date as Day 1, days 1~7 as Week 1, and from day 43 onward as the recovery period. In addition, for the female test animals, the date of administration start was treated as day 0 before mating, the date of mating establishment as day 0 of gestation, and the day of gestation completion as day 0 of lactation.
Positive control:
no
Parental animals: Observations and examinations:
Detailed Symptom Observation
(1) Observation in Home Cage
The animals were observed in the home cage for 1 minute in a resting state.
Examination Items: Tremors, clonic seizures, Tonic convulsions, respiration

(2) Observation during Handling
The animal torsos were gently grasped from the back, and removed from the cage for observation.
Examination Items: Ease of removal from cage, response to handling, aggressiveness, skin (injuries, skin colour), fur (soiling of fur), eyes (eyeball protrusion, closed eyelid state), mucosa (colour of conjunctiva), excreta, lacrimation, ptyalism, piloerection, pupil diameter

(3) Observation in Open Field
Observation was performed for 2 minutes with the animals in a rested state in the centre of an open field (Before placing the animals in the open field, wipe the floor with strongly-wrung wet cloth).
Examination Items: Standing, awareness, urination, defecation, posture/stance, respiration, motor coordination, gait abnormalities, tremors, clonic seizures, Tonic convulsions, normal behaviour, abnormal behaviour

Functional Examination
(1) Responsiveness to Stimuli
This was examined in an open field.
Examination Items: Response to approach, response to contact, auditory response, tail-pinch response, airborne righting reflex.

(2) Grip Strength Measurement
This was measured with a digital force gauge (Type: DPS-5).
Examination Items: Foreleg grip strength, hind leg grip strength

Measurement of Spontaneous Movement
A spontaneous movement measurement device (SUPERMEX, Muromachi Kikai Co., Ltd.) was used. Both male and female animals were moved to a polycarbonate cage (265W×426D×200H mm, Tokiwa Kagaku, K.K.) after the completion of the post-administration observation of week 6 (day 41), and cage domestication was performed (individual care). This was replaced with a new polycarbonate cage prior to measurement, and measurement performed for 1 hour. Furthermore, the measurements were totalled every 10 minutes from the start of measurement.

Body Weight
The male testing animals and male recovery animals were measured on days 1, 8, 15, 22, 29, 36, and 43, and the recovery animals further measured on days 50 and 56. The female satellite animals were measured at the same frequency as the male recovery animals. The female test animals were measured on days 0, 7, and 14 before mating, the mated females on days 0, 7, 14, and 20 of gestation, and the pregnant females on days 0 and 4 of lactation. The measurement used an electronic platform scale (EB-3200S: Shimadzu Corporation, PB3002-S: Mettler-Toledo International, Inc.).

Feed Intake
The male test animals and male recovery animals were measured on days 1~8, 8~15, 22~29, 29~36, 36~40, and 43~50, and the female satellite animals were measured on days 1~8, 8~15, 15~22, 22~29, 29~36, 36~42, 43~50, and 50~56. The female test animals were measured at the same frequency as the body weight measurement. However, measurement was not performed while cohabiting in the mating period (neither males nor females were measured as all cases were mating on days 15~22). The measurement of males after mating with a female had been confirmed was started from the nearest measurement date. The measurement used an electronic platform scale (EB-3200S: Shimadzu Corporation, PB3002-S: Mettler-Toledo International, Inc.). All measured the tared weight on a per-cage basis, and the average daily food intake was calculated per-animal for each measurement period.

Haematological Testing
All surviving animals were fasted (18~24 hours) on day 42 for the male test animals, day 56 of the male recovered animals and female satellite animals, and on day 4 of lactation for the female test animals. 5 male test animals in order from smallest animal number, all the male recovery animals and female satellite animals, and 5 female test animals with early parturition dates in order from smallest animal number were selected as measurement subject animals (blood sampling animals).
On the planned day of dissection the day after fasting, animals were anesthetized by intra-abdominal administration of sodium pentobarbital (Nembutal Injection Fluid, Sumitomo Dainippon Pharma Co., Ltd.), and blood drawn from the Caudal Vena Cava. The following items were tested using the sampled blood. Measurements (9) and (10) used 3.2 w/v% trisodium citrate aqueous solution as the anti-coagulation agent, and the plasma obtained through centrifuging at 12,000 rpm (maximum centrifugal acceleration approx. 12,000g) at 4°C, for 3 minutes was used. Measurement of the other items used blood treated with EDTA-2K anticoagulation agent. Remaining samples were discarded after testing completion.
7.10.6 Blood Biochemistry Testing
Some of the blood collected during the planned dissection, after setting for 30 minutes or more at room temperature and shielded from light, was centrifuged at 3,000 rpm (maximum centrifugal acceleration 1,600g) at approximately 4°C for 10 minutes, and the serum obtained used for measurement of the following items. Measurements were made on the day of blood extraction for all females and males of the blood extraction animals. The remaining serum was stored in a freezer at around -80°C (Allowable range: -60°C or lower), and disposed of by the end of the testing.
Oestrous cyclicity (parental animals):
The oestrus cycle was examined for the female test animals of each group by sampling of vaginal smears during the AM hours from the date of administration start until the start of mating, and the average number of days of oestrus cycle as well as the onset rate of abnormal oestrus period animals were calculated.
Sperm parameters (parental animals):
not examined
Litter observations:
For dams, the ovaries and uterus were extracted during autopsy, and the number of corpora lutea and number of implantations were examined. In addition, the non-pregnant females were likewise examined, and if gross implantations were seen, the uterus was then soaked in a 10vol% ammonium sulphide aqueous solution, and the presence of implantations checked for. As a result, implantations were not seen in 2 cases (Nos. 50107, 50305), and these were treated as non-gravid females.

Observation of Neonates: The number of pups delivered (no. of pups delivered live, no. of stillbirths), sex, and presence of apparent abnormalities were examined on lactation day 0. Thereafter the general condition and presence of deaths were observed each day.
All surviving pups were weighed on an individual basis on days 0 and 4 post-partum. The measurement used an electronic platform scale (PB3002-S: Mettler-Toledo International, Inc.).
Postmortem examinations (parental animals):
Organ Weights
Among the planned dissection animals, 5 were selected from each group in order from smallest animal number in the male test animals, all the male recovery animals and female satellite animals, and five with early parturition dates in order from smallest animal number in the female test animals, and the weights measured for the following organs (bilateral organs were measured together). The testes and epididymides are considered accurate indicators of reproductive and developmental toxicity, and therefore these were measured in all cases. The measurements used an electronic scale (AW120L Shimadzu Corporation). In addition, the weights were measured on the day of dissection, and the relative weights (ratio to body weight) were calculated based thereon. Organ weights were not measured for non-pregnant females.
Brain, heart, liver, kidneys, adrenals, thymus, spleen, testes, epididymides

Pathological Dissection Examination
The animals subject to blood testing following blood sampling, and the non-subject animals, had their abdominal aortas severed, and were desanguinated and euthanized, under anaesthesia according to the method of Item 7.10.5, and autopsied. Non-pregnant animals were euthanized by the above method on day 26 after confirmation of mating, and then were likewise autopsied.

Pathohistological Examination
The following organs and tissues were sampled for all animals, fixed in 10vol% neutral phosphate-buffered formalin fluid, and stored.
Brain, pituitary, thymus, lymph nodes (low jaw/mesenteric), trachea, lungs, intestines (duodenum, jejunum, ileum, cecum, colon, rectum), thyroid/parathyroid (bilateral), heat, liver, spleen, kidneys (bilateral), adrenals (bilateral), bladder, testes (bilateral), epididimides (bilateral), seminal vesicles (including congealing gland), prostatic ventral lobe, ovaries (bilateral), uterus, vagina, bone marrow (right femur), sciatic nerve (right), spinal cord, locations of gross abnormalities
In the control group and 1000 mg/kg group, hematoxylin-eosin stain specimens were fabricated by normal methods from the above organs and tissues for 5 animals in order from the smallest animal number in the male test group, and 5 animals with early parturition date in order from the smallest animal number in the female test animals, the locations of gross abnormalities observed in all animals, including the control group, and the ovaries of 2 females (Nos. 50107, 50305) that were non-gravid even though mating had been confirmed, and then microscopically examined. As a result, no further examinations were performed because no changes thought to have been triggered by the tested substance were observed in the 1000 mg/kg group. Moreover, in one female of the 1000 mg/kg group (No. 50409), it was not possible to check the parathyroid on one side of the sample, therefore only the other side of the sample was examined.
Postmortem examinations (offspring):
On day 4 postpartum, the appearance, including the oral cavity, was examined for all surviving pups, and these were autopsied after having been euthanized in the same manner as the parent animals. Dead pups were autopsied, soaked, and fixed in 10vol% neutral phosphate-buffered formalin fluid, and stored. However, this excluded those that did not bear examination, such as those cannibalized, etc.
Statistics:
yes
Reproductive indices:
(1) Gestation Period: Period from gestation day 0 until day of parturition completion
(2) Birth rate (%): (No. of Females Delivering Live Pups/No. of Conceiving Females) ×100
(3) Implantation index (%): (No. of Implantations/No. of Corpora Lutea) ×100
(4) Delivery dam index (%): (Total No. of Delivered Pups/No. of Implantations) ×100
Offspring viability indices:
(1) Live birth rate (%): (No. of Live Pups Delivered/Total No. of Pups Delivered) ×100
(2) 4-day Survival Rate of Neonates (%): (No. of Live Pups at Lactation Day 4/ No. of Live Pups Delivered) ×100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
See details in section 7.5.1.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
See details in section 7.5.1.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
See details in section 7.5.1.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
See details in section 7.5.1.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
See details in section 7.5.1.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
See details in section 7.5.1.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
See details in section 7.5.1.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No changes thought triggered by the tested substance were seen.
In the measurement of the organ weights, although low adrenal weights were seen in males, and elevated liver weights and low thymus weights in females, histological changes associated with the weight changes were not seen. In addition, no marked changes were seen in the ovaries of the 2 females where mating was confirmed but were non-gravid. The subdermal tumour found in 1 female (No. 50401) was histologically diagnosed as a mammary adenocarcinoma, but this was determined to be a coincidental change from the onset state.
In addition, various histological changes were seen in each group, including the control group. However, the onset of these was not specific to rats, and no apparent correlation was seen with the dose in the onset conditions. We therefore determined that the changes were not toxicologically significant.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
In the oestrus cycle examination, there were no changes in the mean number of oestrus cycle days, and most of the animals of all the groups showed a cycle of 4 or 5 days, and no prolongation or shortening of the oestrus cycle due to the tested substance were seen. Furthermore, although one animal in the control group (No. 50110) exhibited an oestrus cycle disturbance where the dormancy period continued for 11 days, however mating with a male was confirmed, and conception was also confirmed.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No significant differences were seen between the control group and the tested substance dosing groups in mating results, copulation index, number of days needed for mating, number of oestrus periods avoided until mating established, as well as the conception rate. Furthermore, 1 female (No. 50107) that was non-gravid even though mating had been confirmed was seen in the control group, and one (No. 50305) in the 200 mg/kg group. Although no abnormalities were seen in the pathological examination was seen in these females or their counterpart males (Nos. 00107, 00305), and the cause of the non-gestation was unclear, we determined this to be a coincidental change due to the onset conditions.
No significant differences were seen between the control group and the tested substance dosing groups in any of the gestation period, number of corpora lutea, number of implantations, implantation index, birth rate, and delivery dam index. In addition, no abnormalities in parturition and lactation behaviour were seen in any of the dams.
Dose descriptor:
NOAEL
Remarks:
(parental systemic toxicity)
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
Dose descriptor:
NOAEL
Remarks:
(reproductive 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
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Congruent values were seen in the control group and tested substance dosing groups in any of number of pups born, number of live pups delivered, sex ratio, live birth rate and 4-day survival rate of neonates, and no changes triggered by the tested substance were seen. Furthermore, in general condition and external appearance examination as well, no abnormalities triggered by the tested substance were seen in the neonates of any of the groups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Congruent values were seen in the control group and tested substance dosing groups in any of number of pups born, number of live pups delivered, sex ratio, live birth rate and 4-day survival rate of neonates, and no changes triggered by the tested substance were seen. Furthermore, in general condition and external appearance examination as well, no abnormalities triggered by the tested substance were seen in the neonates of any of the groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Both males and females exhibited largely congruent body weights in the control group and the tested substance dosing groups, and no significant difference were seen.
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 abnormalities triggered by the tested substance were seen in autopsy of the surviving pups at 4 days postpartum, as well as autopsy of the deceased pups.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
Critical effects observed:
no
Conclusions:
In addition, the NOEL and NOAEL for reproductive and developmental toxicity was 1000 mg/kg/day in both the parent animals and pups as no changes triggered by the tested substance were observed in the parent animals and pups.
Executive summary:

Copper dibutyldithiocarbamate was administered at doses of 40, 200, and 1000 mg/kg to male and female SD-series rats (Crl:CD(SD)) starting from 14 days prior to mating, and throughout mating, for a total of 42 days for the males, and for the females throughout gestation and parturition up to the 4th day of lactation, and we studied the repeat dosing toxicity and reproductive/developmental toxicity, as well as the recoverability of these changes. The number of animals in a single group was 12 males (including recovered animals), and 12 females (5 animals added to the control group and 1000 mg/kg group as recovered animals), respectively, and in the control group we administered only the medium (0.1% w/v Tween 80-spiked 0.5% w/v CMC-Na aqueous solution).

In general condition observation, faeces including the drug were observed in both males and females in the 1000 mg/kg group (black, tinted with the tested substance). However, no abnormal content was seen in the intestinal tract during autopsy, and histological abnormalities were not seen in the intestinal tract and other organs/tissues as well. It was therefore determined that these changes were triggered by the colour of the tested substance, and that the changes were not toxicologically significant.

The above changes all disappeared in the 2-week recovery period, and reversibility was seen.

Moreover, no changes triggered by the tested substance were seen in the body weight, feed intake amount, behaviour examination, functional examination, and spontaneous movement measurement, or in the haematological analysis as well as urine analysis.

 

No changes triggered by the tested substance were seen in the parent animals in the oestrus cycle, copulation index, conception rate, delivery dam index, gestation period, number of corpora lutea, number of implantations, implantation index, birth rate, or observations of parturition status and lactation.

In examinations of the neonates, no changes triggered by the tested substance were observed in the number of pups born, number of pups born live, sex ratio, live birth rate, and 4-day survival rate of the neonates, nor in the general condition, appearance examination, body weight, or autopsy. Therefore, it was thought that the tested substance did not affect the generation or development of the next generation.

 

Therefore, the no-observed effect level (NOEL) with regard to the repeat dose toxicity under the conditions of this test was 200 mg/kg/day in the males, and 40 mg/kg/day in the females, because elevated total cholesterol was seen in males in the 1000 mg/kg group, and high liver weights for females in the 200 mg/kg group, and the no-observed adverse effect level (NOAEL) to be 1000 mg/kg/day in both males and females because no abnormalities associated with the changes stated above were seen in the pathohistological examination.

 

In addition, the NOEL and NOAEL for reproductive and developmental toxicity was 1000 mg/kg/day in both the parent animals and pups as no changes triggered by the tested substance were observed in the parent animals and pups.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
7
Species:
rat
Quality of whole database:
This OECD 421 study is a reliable study with a klimisch score of 1.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Screening test for reproduction (OECD 421, 2012) :

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 4 post 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.

Screening reproduction study (OECD 422, 2009) :

Copper dibutyldithiocarbamate was administered at doses of 40, 200, and 1000 mg/kg to male and female SD-series rats (Crl:CD(SD)) starting from 14 days prior to mating, and throughout mating, for a total of 42 days for the males, and for the females throughout gestation and parturition up to the 4th day of lactation, and we studied the repeat dosing toxicity and reproductive/developmental toxicity, as well as the recoverability of these changes. The number of animals in a single group was 12 males (including recovered animals), and 12 females (5 animals added to the control group and 1000 mg/kg group as recovered animals), respectively, and in the control group we administered only the medium (0.1% w/v Tween 80-spiked 0.5% w/v CMC-Na aqueous solution).

In general condition observation, faeces including the drug were observed in both males and females in the 1000 mg/kg group (black, tinted with the tested substance). However, no abnormal content was seen in the intestinal tract during autopsy, and histological abnormalities were not seen in the intestinal tract and other organs/tissues as well. It was therefore determined that these changes were triggered by the colour of the tested substance, and that the changes were not toxicologically significant.

The above changes all disappeared in the 2-week recovery period, and reversibility was seen.

Moreover, no changes triggered by the tested substance were seen in the body weight, feed intake amount, behaviour examination, functional examination, and spontaneous movement measurement, or in the haematological analysis as well as urine analysis.

No changes triggered by the tested substance were seen in the parent animals in the oestrus cycle, copulation index, conception rate, delivery dam index, gestation period, number of corpora lutea, number of implantations, implantation index, birth rate, or observations of parturition status and lactation.

In examinations of the neonates, no changes triggered by the tested substance were observed in the number of pups born, number of pups born live, sex ratio, live birth rate, and 4-day survival rate of the neonates, nor in the general condition, appearance examination, body weight, or autopsy. Therefore, it was thought that the tested substance did not affect the generation or development of the next generation.

Therefore, the no-observed effect level (NOEL) with regard to the repeat dose toxicity under the conditions of this test was 200 mg/kg/day in the males, and 40 mg/kg/day in the females, because elevated total cholesterol was seen in males in the 1000 mg/kg group, and high liver weights for females in the 200 mg/kg group, and the no-observed adverse effect level (NOAEL) to be 1000 mg/kg/day in both males and females because no abnormalities associated with the changes stated above were seen in the pathohistological examination.

In addition, the NOEL and NOAEL for reproductive and developmental toxicity was 1000 mg/kg/day in both the parent animals and pups as no changes triggered by the tested substance were observed in the parent animals and pups.

Effects on developmental toxicity

Description of key information

A foetal developmental toxicity study was performed on rats. No adverse maternal or developmental toxicity was observed at the highest dose of 1000 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 October 2017 - 13 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, le Genest-Saint-Isle, France
- Age at the beginning of the treatment period: approximately 10-11 weeks old
- Mean body weight at the beginning of the treatment period: 295 g (range: 235 g to 358 g)
- Fasting period before study: no
- Housing: individually
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 4 or 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 20 November 2017 to 13 December 2017
Route of administration:
oral: gavage
Vehicle:
other: 0.5% (w/w) methylcellulose aqueous solution
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
- Suspension in the vehicle
- Justification for use and choice of vehicle: suitable formulation in the selected vehicle
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle: 10 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: Ultra-Violet-Visible (UV-Vis) spectrophotometric method
Test item concentrations: were within an acceptable range of variation compared to nominal values (+/- 15%) in Weeks 1 and 4.
Homogeneity: homogenous formulations at 10 and 200 mg/mL.
Stability: not assessed, dose formulations were prepared daily.
Details on mating procedure:
- Impregnation procedure: purchased time pregnant
- Proof of pregnancy: vaginal plug (at the breeder's facility); referred to as day 0 post coitum
Duration of treatment / exposure:
Days 6 to 20 post coitum inclusive.
Frequency of treatment:
Daily.
Duration of test:
Day 21 post coitum.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose selection:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous OECD 421 study (reproduction/developmental toxicity screening in rats) where female Sprague-Dawley rats received the test item in 0.5% aqueous methylcellulose by gavage at 10 mL/kg at 50, 250 or 1000 mg/kg/day prior to pairing, during pairing and throughout gestation and lactation until Day 3 post-partum. There were no toxicologically significant effects in females of this study.
Therefore, the same high-dose was used in the present study. The lower doses were selected with an approximately 3-fold interval between each dose.

- Rationale for animal assignment:computerized stratification procedure.
Maternal examinations:
MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends.

CLINICAL SIGNS:
- Time schedule: from arrival, each animal was observed once a day as part of the routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time.

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

POST-MORTEM EXAMINATION:
- Sacrifice on Day 21 post coitum
- Examined: principal thoracic and abdominal organs
Ovaries and uterine content:
The ovaries and uterine content were examined after termination, including:
- Gravid uterus weight
- Number of corpora lutea
- Number of implantation sites
- Number of early and late resorptions
- Number of dead and live fetuses
- Number of uterine scars
- Gross evaluation of placentas
Fetal examinations:
- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: half fetuses per litter
- Skeletal examinations: Yes: half fetuses per litter
- Head examinations: Yes: half fetuses per litter
- Other: fetal weight, fetal sex
Statistics:
Data were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous) or by Fisher’s exact probability test (proportions).
Indices:
% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites
Historical control data:
Cf attached document
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
See table 1.
There were no test item-related clinical signs. Clinical signs observed at 1000 mg/kg/day were of isolated incidence, lasted 1 or 2 days only and were of common background in laboratory rats.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 2.
There were no effects on mean body weight.
At 1000 mg/kg/day and when compared with controls, mean body weight change was slightly lower on Days 6 to 9 p.c. (+13 g vs. +18 g, p<0.01) with a return towards control values thereafter. Therefore, this finding was considered to be test item-related and non-adverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects on mean food consumption.
High food consumptions noted in one control female (on Days 18 to 21 p.c.) and in one female at 300 mg/kg/day (from Days 9 to 18 p.c.) were considered to be related to food spillage which is rather common in laboratory rodents.
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:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
See table 4.
There were no test item-related necropsy findings (isolated incidences, no dose-relationship and/or of common background in laboratory rats).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Uterus weight, carcass weight and net body weight change from Day 6 post coitum:
See table 3.
There were no effects on mean carcass weight, mean gravid uterus weight, or mean net body weight change.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
See table 5.
Number of dams with pre-implantation loss = 17, 15, 10, 16, at 0, 100, 300, 1000 mg/kg/day, respectively
Number of dams with post-implantation loss = 12, 15, 10, 13 at 0, 100, 300, 1000 mg/kg/day, respectively
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See table 5.
No dams with total resorption.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
See table 5.
Number of dams with resorptions = 12, 15, 10, 13 at 0, 100, 300, 1000 mg/kg/day, respectively
Dead fetuses:
no effects observed
Description (incidence and severity):
See table 5.
No dead fetuses.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
At hysterectomy on Day 21 p.c., there were 24/24, 22/24, 23/24 and 24/24 pregnant dams with live fetuses in the groups treated at 0, 100, 300 or 1000 mg/kg/day, respectively.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
See table 6
There were no effects on mean fetal body weight.
Mean male fetal body weight: 6.04, 6.03, 6.04, 5.93 g, at 0, 100, 300 or 1000 mg/kg/day, respectively
Mean female fetal body weight: 5.63, 5.58, 5.62, 5.63 g, at 0, 100, 300 or 1000 mg/kg/day, respectively
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See table 5
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See table 7
There were no effects on mean percentage of male fetuses (sex-ratio).
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
See tables 9 and 14.
External malformations observed at 100 and 300 mg/kg/day were not considered to be test item treatment related (no dose-relationship and most of them recorded at litter incidences comparable to that reported in historical control data).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
See tables 13 and 14.
In test item-treated groups, none of the skeletal malformations were considered to be test item treatment related: they were noted at isolated incidences and there were no findings at 1000 mg/kg/day.


Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
See tables 11 and 14.
The malformations observed at 100 mg/kg/day were considered to be not related to the test item treatment as they were noted in only one fetus and/or found also in Historical Control Data.

Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
External variations
See table 8.
All external variations were considered to be incidental as there was no dose-relationship. The incidences were comparable with controls or isolated, and/or they were of common background in laboratory rats of this strain.

Skeletal examinations
Cartilage
See table 12.
None of the cartilage findings were considered to be test item treatment-related; they were noted with no dose-relationship, at isolated or low incidences, and/or can be found in Historical Control Data.
The other cartilages were present or of normal morphology.

Variations
Fetal skeletal variations were not considered to be test item-related (including the statistically higher number of fetuses with ossification point on 14th thoracic vertebra at 100 mg/kg/day when compared with controls): they were at comparable/lower incidences to/than controls, within historical control data, not dose-related, not statistical significant and/or noted at isolated/low incidence.

Soft tissue variations
See table 10.
These visceral variations were considered to be incidental as there was no dose-relationship, the incidences were comparable with (or lower than) controls, isolated, and/or of common background in laboratory rats of this strain.
Dose descriptor:
NOAEL
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
Abnormalities:
effects observed, non-treatment-related
Developmental effects observed:
no

Table 1: Clinical signs

Dose level (mg/kg/day)

0

100

300

1000

Cutaneous lesion on neck dorsal area

1

0

0

0

Piloerection

0

0

0

1

Reflux at dosing

0

0

0

1

Number of affected animals

1/24

0/24

0/24

2/24

 

Table 2: Body weight and body weight change

Dose level (mg/kg/day)

0

100

300

1000

Mean body weight

 

 

 

 

Day 6 p.c.

295

297

295

296

 

 

+1

0

0

Day 9 p.c.

312

312

310

308

 

 

0

-1

-1

Day 21 p.c.

451

447

446

446

 

 

-1

-1

-1

Mean body weight change

 

 

 

 

Days 6 - 9 p.c.

+18

+16

+15

+13**

Days 9 - 21 p.c.

+139

+135

+137

+138

Days 6 - 21 p.c.

+156

+151

+151

+151

In italic, percentage difference (%) vs. controls,

Statistically significant vs. controls:**: p<0.01.

 

Table 3: Net body weight

Dose level (mg/kg/day)

0

100

300

1000

Gravid uterus weight

102.1

99.5

101.2

97.3

Carcass weight

348.5

347.8

345.0

349.2

Net weight change from Day 6 p.c.

54.0

51.3

49.9

53.5

 

Table 4: Macroscopic post-mortem examination

Dose level (mg/kg/day)

0

100

300

1000

Lymph node(s) (bronchic): enlarged

0

0

1

0

Lungs: Irregular color (reddish)

1

0

0

1

Absence of kidney (right)

1a

0

0

0

Enlarged kidney (left)

1a

0

0

0

Segmental aplasia of uterine horn

1a

0

0

1

Enlarged placenta(s)

0

1

1

1b

Granular placenta

0

0

0

1b

No remarkable observations

22

23

22

21

a:one female ;b: same placenta of one female.

Table 5: Hysterectomy data

Dose level (mg/kg/day)

0

100

300

1000

HCD

Number of females with live fetuses at termination

24

22

23

24

388

Mean number of corpora lutea

14.6

14.9

14.6

14.8

[13.8-16.0]

Mean number of implantation sites

13.2

13.6

13.5

13.2

[12.5-14.5]

Mean pre-implantation loss (%)

9.9

8.1

8.0

10.6

[6.2-14]

Mean number of live fetuses

12.5

12.4

12.6

12.2

[11.6-13.8]

Dead fetuses (%)

0.0

0.0

0.0

0.0

[0.0-0.5]

Mean number of implantation scars

0.0

0.0

0.0

0.0

np

Mean number of early resorptions

0.7

1.1

0.9

1.0

Early+late resoprtions:[0.5-1.35]

Mean number of late resorptions

0.0

0.1

0.0

0.1

Mean post-implantation loss (%)

5.8

9.0

8.1

8.0

[3.5-11.1]

HCD: Historical Control Data (2014-2016; Sprague-Dawley rats): study mean range [min-max]; np: not presented in HCD.

 

Table 6: Fetal body weights

Dose level (mg/kg/day)

0

100

300

1000

Mean fetal body weight (g)

5.84

5.79

5.80

5.80

 

Table 7: Fetal sex

Dose level (mg/kg/day)

0

100

300

1000

Mean percentage of male fetuses (%)

53.6

47.8

49.6

52.1

 

 

Table 8: External variations

Dose level (mg/kg/day)

0

100

300

1000

HCD

Number of litters

24

22

23

24

388

Number of fetuses

300

272

289

292

5000

General

 

 

 

 

 

Local edema, F (L)

0.3 (4.2)a

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.8 (9.5)

Polyhydramnios, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.3 (4.2)

-

Paw and digit

 

 

 

 

 

Malrotated paw, F (L)

0.3 (4.2)a

0.4 (4.5)b

0.0 (0.0)

0.0 (0.0)

0.4 (5.6)

Paw hyperflexion, F (L)

0.3 (4.2)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

-

Litters with external variations, n (%)

2 (8.3)

1 (4.5)

0 (0.0)

1 (4.2)

6 (1.5)

Fetus with external variations, n (%)

2 (0.7)

1 (0.4)

0 (0.0)

1 (0.3)

7 (0.1)

F: fetal incidence, L: litter incidence, n: number.

a: one fetus ,b:anotherr fetus which also had bent tail (see next § Malformations).

HCD: Historical Control Data (2014-2016; Sprague-Dawley rats): fetal maximal incidences (litter maximal incidences) for the findings, -: none in HCD.

 

Table 9: External malformations

Dose level (mg/kg/day)

0

100

300

1000

HCD

Number of litters

24

22

23

24

388

Number of fetuses

300

272

289

292

5000

General

 

 

 

 

 

Anasarca, F (L)

0.0 (0.0)

0.0 (0.0)

0.3 (4.3)c

0.0 (0.0)

-

Mouth, jaw, palate

 

 

 

 

 

Cleft palate, F (L)

1.7 (4.2)

0.0 (0.0)

0.3 (4.3)c

0.0 (0.0)

0.4 (5.0)

Trunk

 

 

 

 

 

Short trunk, F (L)

0.0 (0.0)

0.4 (4.5)a

0.0 (0.0)

0.0 (0.0)

0.4 (5.0)

Anal atresia, F (L)

0.0 (0.0)

0.4 (4.5)a

0.0 (0.0)

0.0 (0.0)

0.4 (5.0)

Omphalocele, F (L)

0.0 (0.0)

0.0 (0.0)

0.3 (4.3)

0.0 (0.0)

0.4 (5.3)

Ombilical hernia, F (L)

0.0 (0.0)

0.0 (0.0)

0.3 (4.3)c

0.0 (0.0)

0.4 (4.8)

Tail

 

 

 

 

 

Bent tail, F (L)

0.0 (0.0)

0.4 (4.5)b

0.0 (0.0)

0.0 (0.0)

0.4 (5.0)

Short tail, F (L)

0.0 (0.0)

0.4 (4.5)a

0.0 (0.0)

0.0 (0.0)

1.0 (4.3)

Litters with external malformations, n (%)

1 (4.2)

1 (4.5)

2 (8.7)

0 (0.0)

11 (2.8)

Fetus with external malformations, n (%)

5 (1.7)

2 (0.7)

2 (0.7)

0 (0.0)

13 (0.3)

F: fetal incidence, L: litter incidence, n: number.

a: one fetus ,b: another fetus,c: another fetus.

HCD: Historical Control Data (2014-2016; Sprague-Dawley rats): fetal maximal incidences (litter maximal incidences) for the findings, -: none in HCD.

Table 10: Soft tissue variations

Dose level (mg/kg/day)

0

100

300

1000

HCD

Number of litters

24

22

23

24

387

Number of fetuses

145

130

138

139

2404

Kidneys

 

 

 

 

 

Dilated renal pelvis, F (L)

0.7 (4.2)

1.5 (9.1)

0.7 (4.3)

1.4 (8.3)

9.5 (28.6)

Vessels

 

 

 

 

 

Absent innominate artery, F (L)

0.7 (4.2)

0.8 (4.5)

0.0 (0.0)

0.0 (0.0)

5.1 (25.0)

Ureter

 

 

 

 

 

Dilated ureter, F (L)

5.5 (20.8)

3.1 (13.6)

1.4 (8.7)

3.6 (16.7)

7.1 (28.0)

Thymus

 

 

 

 

 

Reddish focus, F (L)

0.0 (0.0)

0.0 (0.0)

0.7 (4.3)

0.0 (0.0)

-

Litters with visceral variations, n (%)

5 (20.8)

4 (18.2)

3 (13.0)

4 (16.7)

86 (22.2)

Fetus with visceral variations, n (%)

8 (5.5)

5 (3.8)

3 (2.2)

5 (3.6)

119 (5.0)

F: fetal incidence, L: litter incidence, n: number.

HCD: Historical Control Data (2014-2016; Sprague-Dawley rats): fetal maximal incidences (litter maximal incidences) for the findings, -: none in HCD.

 

Table 11: Soft tissue malformations

Dose level (mg/kg/day)

0

100

300

1000

HCD

Number of litters

24

22

23

24

387

Number of fetuses

145

130

138

139

2404

Mouth, jaw, palate

 

 

 

 

 

Cleft palate, F (L)

2.1 (4.2)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

-

Brain

 

 

 

 

 

Dilated 4thcerebral ventricle, F (L)

0.0 (0.0)

0.8 (4.5)

0.0 (0.0)

0.0 (0.0)

0.7 (4.2)

Gonads

 

 

 

 

 

Absent, F (L)

0.0 (0.0)

0.8 (4.5)a

0.0 (0.0)

0.0 (0.0)

-

Litterswith visceral malformations, n (%)

1 (4.2)

2 (9.1)

0 (0.0)

0 (0.0)

7 (1.8)

Fetuswith visceral malformations, n (%)

3 (2.1)

2 (1.5)

0 (0.0)

0 (0.0)

13 (0.5)

F: fetal incidence, L: litter incidence, n: number.

HCD: Historical Control Data (2014-2016; Sprague-Dawley rats), -: none in HCD.

a: one fetus with external malformations of the trunck/tail.

Table 12: Fetal skeletal examinations

Cartilage

Dose level (mg/kg/day)

0

100

300

1000

HCD

Number of litters

24

22

23

24

388

Number of fetuses

155

142

151

153

2596

Cervical vertebrae

 

 

 

 

 

Bipartite cartilage of centrum, F (L)

0.0 (0.0)

0.7 (4.5)

0.0 (0.0)

0.0 (0.0)

-

Thoracic vertebrae

 

 

 

 

 

Misshapen cartilage of centrum, F (L)

0.6 (4.2)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

-

Bipartite cartilage of centrum, F (L)

0.0 (0.0)

0.7 (4.5)a

0.0 (0.0)

0.0 (0.0)

0.7 (4.5)

Caudal vertebrae

 

 

 

 

 

Less than 15vertebrae, F (L)

0.0 (0.0)

0.7 (4.5)

1.3 (8.7)

0.0 (0.0)

3.0 (8.0)

Misshapen cartilage, F (L)

0.0 (0.0)

0.7 (4.5)a

0.0 (0.0)

0.0 (0.0)

-

Sternebrae

 

 

 

 

 

Bipartite cartilage, F (L)

0.0 (0.0)

0.0 (0.0)

0.7 (4.3)c

0.0 (0.0)

-

Rib

 

 

 

 

 

Absent cartilage, F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.7 (4.2)

5.1 (21.7)

Fused cartilages, F (L)

0.0 (0.0)

0.7 (4.5)a

0.0 (0.0)

0.0 (0.0)

0.6 (4.3)

F: fetal incidence, L: litter incidence, n: number.

HCD: Historical Control Data (2014-2016; Sprague-Dawley rats): fetal maximal incidences (litter maximal incidences) for the findings, -: none in HCD.

a: one fetus with the malformation supernumerary lumbar vertebra,c: another fetus.


 

Table 13: Fetal skeletal malformations

Dose level (mg/kg/day)

0

100

300

1000

HCD

Number of litters

24

22

23

24

388

Number of fetuses

155

142

151

153

2596

Palate

 

 

 

 

 

Split, F (L)

1.3 (4.2)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.7 (5.0)

Cervical vertebrae

 

 

 

 

 

Cervical rib, F (L)

0.0 (0.0)

0.0 (0.0)

0.7 (4.3)

0.0 (0.0)

-

Lumbar vertebrae

 

 

 

 

 

Supernumerary , F (L)

0.0 (0.0)

0.7 (4.5)b

0.0 (0.0)

0.0 (0.0)

-

Sternebrae

 

 

 

 

 

Fused, F (L)

0.0 (0.0)

0.0 (0.0)

0.7 (4.3)a

0.0 (0.0)

1.3 (8.7)

Litterswith skeletal malformations, n (%)

1 (4.2)

1 (4.5)

2 (8.7)

0 (0.0)

15 (3.9)

Fetuswith skeletal malformations, n (%)

2 (1.3)

1 (0.7)

2 (1.3)

0 (0.0)

18 (0.7)

F: fetal incidence, L: litter incidence, n: number.

HCD: Historical Control Data (2014-2016; Sprague-Dawley rats): fetal maximal incidences (litter maximal incidences) for the findings, -: none in HCD.

a: one fetus,b: another fetus.

 

 

Table 14: Malformed fetuses

Dose level (mg/kg/day)

0

100

300

1000

Number of litters

24

22

23

24

Number of fetuses with external examination

300

272

289

292

Number of fetuses with visceral examination

145

130

138

139

Number of fetuses with skeletal examination

155

142

151

153

 

L29393 -01, -02, -04, -10, -13: cleft/split palate

L29418-09: short trunk, anal atresia, short tail, absent gonads

L29458-05: anasarca, cleft palate, umbilical hernia, fused sternebrae

 

 

 

 

L29418-01: bent tail

 

 

 

 

 

L29431-02: dilated 4thcerebral ventricle

 

L29419-07: supernumerary lumbar vertebra

L29443-01: omphalocele

 

 

 

L29449-07: cervical rib

 

Litters with malformations, n (%)

1 (4.2)

3 (13.6)

3 (13.0)

0 (0.0)

Fetuses with malformations, n (%)

5 (1.7)

4 (2.2)

3 (1.4)

0 (0.0)

 

Conclusions:
Under the experimental conditions and results of this study, the No Observed Adverse Effect Level (NOAEL) for maternal parameters and for embryo-fetal development was considered to be 1000 mg/kg/day in absence of adverse test item treatment effects at this dose level.
Executive summary:

The objective of this GLP study was to evaluate the potential toxic effects of the test item, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy [Days 6 to 20 post-coitum (p.c.) inclusive].

 

Methods

Three groups of 24 time-mated Sprague-Dawley rats were administered the test item, daily by gavage from Days 6 to 20 p.c. inclusive at 100, 300 or 1000 mg/kg/day as a suspension in the vehicle [0.5% (w/w) methylcellulose aqueous solution]. A dose volume of 10 mL/kg/day was used. One additional group of 24 females received the vehicle alone under the same experimental conditions.

Test item concentration was checked two times in formulations given to the animals.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals.

On Day 21 p.c., surviving females were euthanized and submitted to a macroscopic post-mortem examination. Hysterectomies were performed and the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal/cartilage abnormalities.

 

Results

The test item concentrations determined in Weeks 1 and 4 remained within an acceptable range of -10.5% to +0.8% when compared to the nominal values (± 15% required). No test item was observed in the control dose formulations.

 

At hysterectomy on Day 21 p.c., there were 24/24, 22/24, 23/24 and 24/24 pregnant dams with live concepti in the groups treated at 0,100, 300 or 1000 mg/kg/day, respectively.

 

There were no test item treatment-related effects in the dams in terms of mortality, clinical signs, necropsy findings, mean body weight, mean food consumption, mean carcass weight, mean gravid uterus weight, mean net body weight change from Day 6 p.c., and mean hysterectomy data. Mean body weight change was slightly lower at 1000 mg/kg/day on Days 6 to 9 p.c.(+13 g vs. +18 g in controls, p<0.01); this was considered to be test item-related and non-adverse.

 

There were no test item treatment-related effects on sex ratio or mean fetal body weight and no test item treatment-related finding at fetal examination (external, visceral and skeletal variations or malformations).

 

Conclusion

Under the experimental conditions and results of this study, the No Observed Adverse Effect Level (NOAEL) for maternal parameters and for embryo-fetal development was considered to be 1000 mg/kg/day in absence of adverse test item treatment effects at this dose level.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
7
Species:
rat
Quality of whole database:
The study is considered to be reliable, following the test guideline and GLP requirements.
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

Developmental toxicity study in rats (Bentz 2018):

The objective of this GLP study was to evaluate the potential toxic effects of the test item, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy [Days 6 to 20 post-coitum (p.c.) inclusive].

Three groups of 24 time-mated Sprague-Dawley rats were administered the test item, daily by gavage from Days 6 to 20p.c.inclusive at 100, 300 or 1000 mg/kg/day as a suspension in the vehicle [0.5% (w/w) methylcellulose aqueous solution]. A dose volume of 10 mL/kg/day was used. One additional group of 24 females received the vehicle alone under the same experimental conditions.

At hysterectomy on Day 21p.c., there were 24/24, 22/24, 23/24 and 24/24 pregnant dams with live concepti in the groups treated at 0,100, 300 or 1000 mg/kg/day, respectively.

There were no test item treatment-related effects in the dams in terms of mortality, clinical signs, necropsy findings, mean body weight, mean food consumption, mean carcass weight, mean gravid uterus weight, mean net body weight change from Day 6p.c., and mean hysterectomy data. Mean body weight change was slightly lower at 1000 mg/kg/day on Days 6 to 9p.c.(+13 gvs.+18 g in controls, p<0.01); this was considered to be test item-related and non-adverse.

There were no test item treatment-related effects on sex ratio or mean fetal body weight and no test item treatment-related finding at fetal examination (external, visceral and skeletal variations or malformations).

Under the experimental conditions and results of this study, the No Observed Adverse Effect Level (NOAEL) for maternal parameters and for embryo-fetal development was considered to be 1000 mg/kg/day in absence of adverse test item treatment effects at this dose level.

Justification for classification or non-classification

Based on the available data, no classification for reproduction toxicity is required for the registered substance according to the Regulation EC 1272/2008.