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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:
2011-02-03 until 2011-04-12.
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
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
27 July 1995
Deviations:
no
Remarks:
No deviation on the performance of the main study. An additional satellite group was treated for 28 days to assess organ toxicity.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
ammonium 2,2-difluoro-2-{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate
EC Number:
682-238-0
Cas Number:
1190931-27-1
Molecular formula:
C6H4F9NO6
IUPAC Name:
ammonium 2,2-difluoro-2-{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate
Details on test material:
Name : cC6O4
Alternative name : C6O4 cyclic
Batch number : Lotto 1+lotto 2 ac3
Purity : 46.09% w/w of water
Storage at RTC : Ambient condition
Description at first use : Yellowish liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
A total of 100 Hsd: Sprague Dawley SD rats (50 males and 50 virgin females), 6 to 7 weeks old and weighing 176 to 200 g for males and 151 to 175 g for females, were obtained by Harlan Italy S.r.l., San Pietro al Natisone (UD), Italy.
Body weight ranges were 182 to 201 g for males and 155 to 166 g for females. An acclimatisation period of 19 days was allowed before the start of treatment.

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. 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.
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. 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. Suitable nesting material was provided and changed at least 3 times a week.
Drinking water was supplied ad libitum to each cage via water bottles.
A commercially rodent diet 4 RF 21 was offered ad libitum.

On the day of allocation (6 days prior to the start of treatment) all animals were weighed. Animals at the extremes of the weight distribution and some animals showing minor signs of ill health were excluded to leave the required number of animals. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.
Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed up to 5 of one sex per cage.
The cages were identified by a label recording the study number, animal numbers and details of treatment.
The arrangement of cages in batteries was such that cages from each treatment group was evenly distributed across the battery to minimise possible environmental effects.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified water
Details on exposure:
The test item was administered orally by gavage at a dose volume of 10 ml/kg body weight.
Control animals received the vehicle alone at the same dose volume.
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 the animal which did not have positive identification of mating after 14 days of pairing was changed within its treatment group. The subsequent pairing was monitored for mating as described above until indications of pregnancy were evident.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males (Main groups): Male animals of the main groups were dosed for 2 consecutive weeks prior to pairing and thereafter through to the day before necropsy, for a total of 6 consecutive weeks.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females (Main groups): Female animals of the main groups were dosed for 2 consecutive weeks prior to pairing and thereafter during gestation and post partum periods up to Day 3 post partum (including the day of parturition), with the exception of Group 4 dams, which were sacrificed between Day 0 and Day 1 post partum, due to total litter loss.
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.
Males and Females (Satellite group): Animals of the satellite group were dosed for a minimum of 28 consecutive days.
Frequency of treatment:
once a day, 7 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
Males / dry salt
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Males / dry salt
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
Males / dry salt
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
Females /dry salt
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Females /dry salt
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
Females / dry salt
Dose / conc.:
0.5 mg/kg bw/day (nominal)
Remarks:
Males & females/dry salt. Satellite groups for systemic organ toxicity assessment (liver)
No. of animals per sex per dose:
Groups of 10 males received the test item, by gavage at dosages of 5, 20 and 60 mg/kg/day (dry salt) and groups of 10 females received the test item at dosages of 5, 20 and 80 mg/kg/day (dry salt).
One satellite group for liver toxicity evaluation, comprised 5 male and 5 female animals and was treated at 0.5 mg/kg/day (dry salt).
Control animals:
yes
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on the previous 28-day study (RTC Study no. 82340), performed at 5, 20 and 60 mg/kg/day in the males and 20, 60 and 200 decreased to 100 mg/kg/day by Week 3, in the females. The dose level of the satellite group was selected in order to assess the NOAEL, not found in the previous study due to the presence of toxic effects in the liver at all the dose levels investigated.

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement 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: Yes
- Time schedule for examinations: Males (Main and Satellite groups) and Females (Satellite group) were weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy. Females (Main groups) 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 1 post partum).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

ORGAN WEIGHTS:
In males of the main groups: epididymides and testes
In the females of the main groups: ovaries
In males and females of the satellite group: liver

ORGANS FIXATION:
reproductive organs from all animals of the main groups. Liver from the animals of the satellite group.

HISTOPATHOLOGY:
Study list of organs/tissues for histopathological examination (see below)
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 following:
a) anomalies of the oestrous cycle;
b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Sperm parameters (parental animals):
Parameters examined in male parental generation:
- testis weight, 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 surviving pups were also weighed on Day 4 post partum. All pups found dead were necropsied.
Postmortem examinations (parental animals):
All females killed at term (including 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).
Postmortem examinations (offspring):
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 were assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables were 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 were carried out by means of the nonparametric Kolmogorov-Smirnov test (main groups only).
Reproductive indices:
Male Fertility index (%) = (number of males which induced pregnancy/Number of males paired)*100
Female Fertility index (%) = (number of pregnant females/number of females paired)*100
Copulatory interval: mean number of days between pairing and mating
Copulatory index (%) = (number of animals mated/number of animals paired)*100
Offspring viability indices:
Pre-birth loss: (Nb of visible implantations - total litter size)*100 / Nb of visible implantation
Pup loss at birth: (total litter size - liver litter size)*100 / total litter size
Cumulative pup loss on day 4
Sex ratios calculated at birth and on day 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No signs of toxicological significance were observed in male animals from the control, low and mid-dose groups.
Hunched posture, piloerection and an emaciated appearance were observed in the majority of the high dose males (receiving 60 mg/kg/day), generally from the end of the second week of treatment.
No significant clinical signs were seen in female animals from the control, low and mid-dose groups. Piloerection and rales were observed in individual high dose females (receiving 80 mg/kg/day).
No clinical signs were observed in the satellite group animals.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of males were similar between groups during the pre-mating phase of the study. Reductions in body weights (from -12% on Day 8 to -28% on Day 22) were seen in the high dose males from pairing to sacrifice and in the high dose females (from -8% to -20%) during post coitum period when compared to controls.
Reduced body weight gain/body weight loss was observed in the high dose males during pairing up to sacrifice. Terminal body weight showed a statistically significant reduction in the mid- and high dose males (-8% and -30%).
Statistically significant reductions in body weight gain (-45%, -76% and -36% on Days 7, 14 and 20, respectively) were seen the high dose females during the gestation period.

In the satellite group treated for 28 days at 0.5 mg/kg/day, animals gained weight over the entire period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No effects on food consumption were observed in the males and females during the pre-pairing period.
Reduced food consumption was observed in the high dose females during the post-coitum period. (Female data table)
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reductions were seen in the high dose females during the post-coitum period, consistent with reduced body weight and body weight gain.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic evaluation was performed on all animals from the control and high dose groups, liver in the satellite groups and all gross abnormalities from all animals.
As potential treatment-related changes were noted in coagulating gland, seminal vesicle and prostate in high dose males and a reduced number of corpora lutea (ovaries) was observed in high dose females, the histopathological examination was extended to the above mentioned organs in the intermediate dose groups (mid-dose group - 20 mg/kg/day and low dose group - 5 mg/kg/day). In addition, histopathological examination was also extended to prostate gland, seminal vesicles and coagulating glands in all control animals.

Main groups
Treatment-related findings were found in the liver, ovaries, coagulating glands, seminal vesicles, prostate and thymus.
The following are details of the treatment-related lesions seen in the high dosed animals.
Specific notes are added in those organs in which a treatment-related effect was observed in the intermediate and low dose groups.
LIVER: treatment-related changes seen in the high dose included diffused minimal to moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia. This was associated with the presence of minimal to mild inflammatory cell foci, single cell hepatocytic necrosis and multifocal hepatocytic necrosis, minimal multifocal accumulation of brownish pigment-laden macrophages, minimal multifocal bile duct hyperplasia and mild nodular hyperplasia. In the intermediate male group (i.e., Group 3), minimal to moderate diffused hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia, was noted. This was occasionally associated with the presence of minimal single cell hepatocytic necrosis.
THYMUS: treatment-related changes seen in the high dose female included diffused minimal to marked cortical atrophy, resulting from loss of the lymphocytes. This change was usually associated with multifocal lymphocytic necrosis. In a single male rat from Group 2, minimal congestion was noted.
COAGULATING GLAND, SEMINAL VESICLE, PROSTATE: treatment-related changes seen in the high dose included diffused minimal to mild reduced secretion, associated with minimal to mild atrophy of the lining glandular epithelium.
OVARIES: treatment-related changes seen only in high dose indicated mild reduction in the number of corpora lutea when compared to the controls.

All other changes are not considered as related to treatment. In particular, a singular case of monolateral testicular atrophy or absence of sperm and fibrosis in epididymides was observed in a low dose group animal. Such changes are known to occur spontaneously in untreated Sprague Dawley rats of the same age or under our experimental conditions.

Satellite group: No treatment related changes were seen at the histopathological examination.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No significant differences in oestrus cycle, pre-coital interval and copulation plugs were observed between treated and control animals.
Reproductive parameters (copulatory index and fertility index) did not show intergroup differences in neither sex.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Absence of sperm and fibrosis in epididymides was observed in a low dose group animal and was considered incidental.
Reproductive performance:
no effects observed
Description (incidence and severity):
No significant differences in pre-coital interval and copulation plugs were observed between treated and control animals.
Reproductive parameters (copulatory index and fertility index) did not show intergroup differences in neither sex.

All females were pregnant. Unilateral implantation was recorded in 1 female of the control group. All females mated, even though mating was not detected in 1 mid-dose female.

Reductions in the number of implantation and corpora lutea (-17% and -13%, respectively, although not statistically significant) were observed in the high dose dams (80 mg/kg/day). Total litter size was significantly reduced in these animals. Due to the mortality or cannibalisation of all pups between Days 0 and 1, pre-birth loss number and percentage were not calculable in Group 4 animals.
No differences in the number of implantation or pre-birth loss data were noted in the other treated animals compared to controls.
No significant differences in gestation length were observed. The slight reduction observed in animals of Group 3 was not considered of toxicological relevance.

Details on results (P0)

Males were more sensitive to liver effects than females (effects observed at a lower dose).
In males of the high dose (60 mg/kg/day), effects consisted of reduced body weight, Increased relative weights of testes, various histopathological findings in the liver (including hepatocytic hypertrophy, inflammation, hepatocytic necrosis, bile duct hyperplasia). Treatment-related findings were found in the coagulating glands, seminal vesicles, and prostate. Milder effects were noted in the intermediate dose group.
In females of the high dose group (80 mg/kg/day), effects consisted of reduced body weight, reduced food consumption during post-coitum period; macroscopic changes in the liver and thymus, histopathological findings in the liver, thymus, and ovaries. Females of this group had a lower number of corpora lutea. Iin addition, there was a reduction in the number of implantations and corpora lutea, and a peri-natal litter loss.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
80 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
other: females
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
60 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
other: males
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Pre-weaning clinical signs observed in the pups during the lactation period showed small pups, often showing an apparent absence of food intake, with scabs or bruises on the muzzle. The incidence of these signs increased with the dose level of dams.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The number of females with live pups on Day 4 post partum was 10 in each of the control, low dose and mid-dose groups. Total litter loss or cannibalisation during parturition were observed on Days 0 or 1 post partum in the high dose dams which were sacrificed.

All pups of high dose dams were found dead or were cannibalised between Days 0 and 1. Those which could be observed showed an apparent absence of food intake.
Necropsy examination of the early decedent pups revealed the absence of milk in the stomach in the majority of pups from all dose groups. Autolysis was seen in a number of pups from all dose groups.
No significant abnormalities were recorded in the pups sacrificed on Day 4 post partum.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects were observed on the mean total and individual litter weights in the 2 lowest dose groups.
The high dose group could not be assessed due to total litter loss in all dams.
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
All pups of high dose dams were found dead or were cannibalised between Days 0 and 1. Those which could be observed showed an apparent absence of food intake.
Necropsy examination of the early decedent pups revealed the absence of milk in the stomach in the majority of pups from all dose groups. Autolysis was seen in a number of pups from all dose groups.
No significant abnormalities were recorded in the pups sacrificed on Day 4 post partum.

Details on results (F1)

All pups from Group 4 litters (dams dosed at 80 mg/kg/day) were found dead or were cannibalised during parturition or between the day of parturition (Day 0) and Day 1. Those which could be observed showed an apparent absence of food intake.
Total litter size showed a significant reduction in animals dosed at 80 mg/kg/day, when compared to controls (-50%). Live litter size of the high dose group was also consequently severely reduced on Day 0, with total litter loss recorded on Day 1 post partum. The mortality or cannibalisation of all pups from the high dose dams did not allow an evaluation of mean total and individual litter weights or the sex ratio.

Total litter size, live litter size and pup loss were comparable between Groups 2, 3 (dosed at 5 and 20 mg/kg/day, respectively) and controls on Days 1 and 4 post partum. A slight but not statistically significant reduction (-8%) in mean pup weight was seen in animals from mid-dose groups (20 mg/kg/day). This slight reduction was not considered to be of toxicological relevance as, in the majority of cases, parturition occurred on Day 21.
Sex ratio (as % of males) and the total number of pups were comparable between Groups 2 and 3 and controls on Days 1 and 4 post partum.

Since the indicated effects on litters noted in the high dose females were observed in the presence of a marked maternal toxicity (severe reductions in body weight and body weight gain, severe changes in the liver and thymus) a correct evaluation/interpretation of the developmental effects could not be done at this dose level.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
other: At the high dose, smaller litter size due to perinatal loss or cannibalism during parturition or between day 0 and 1.

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
80 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects

Any other information on results incl. tables

Since the indicated effects on pregnancy noted in the high dose females were observed in the presence of a marked maternal toxicity a correct evaluation/interpretation of the reproductive effects could not be done at this dose level.

Reproduction data - summary

 Dose (mg/kg/day)  0 5  20  80  
 Females paired, N  10 10  10  10  
 Females mated, N  10  10  10  10  
 Pregnant females, N  10  10  10  10  
 Mating index, %  100  100  100  100  
 Fertility index, % 100   100  100  100  
 Mean pre-coital time, days  5.9 + 4.5  3.2 + 1.5  3.7 + 2.4  4.4 + 3.6  
 Mean corpora lutea, N  15.80 + 3.61  15.60 + 1.71  15.90 + 2.56  13.70 + 2.83  
 Mean implantation sites, N  15.50 + 3.84  15.20 + 2.25  15.80 + 2.49  12.80 + 4.02  
 Gestation length, days  22.00 + 0.00  21.80 + 0.42  21.44 + 0.53  21.90 + 0.57  
 Litter number, N  10  10  10  (6)  high dose: ND due to cannibalism at parturition
 Mean total litter size, N  14.60 + 4.17  14.90 + 2.28  14.80 + 2.78  °  
Mean pre-birth loss, N  0.90 + 0.74  0.30 + 0.48  1.00 + 1.05  °  
 Pre-birth loss, %  6.59 + 5.94  1.97 + 3.17  6.54 + 7.06  ND  

° not included in statistical analysis

ND not determinable

Litter data - summary (groups 1 to 3)

 Dose (mg/kg/day)  0  5  20
 Litter number, N  10  10  10
 Total litter size at birth, mean  14.60 + 4.17  14.90 + 2.28  14.80 + 2.78
 Live pups at birth, mean  14.30 + 4.08  14.60 + 2.12  14.60 + 2.76
Pup loss, %   1.88 + 4.04  1.86 + 3.01  1.36 + 2.95
 Litter affected, N  2  3  2
       
 Total litter size at birth, N  146  149  148
 Total live pups at birth, N  143  146  143
 Day 1 post-partum:      
 Mean litter weight day 1 p.p., g  84.81 + 26.72  89.70 + 12.73  82.54 + 14.19
 Mean pup weight day 1 p.p., g  6.38 + 0.25  6.18 + 0.46  5.84 + 0.53
 Day 4 post-partum:      
 Mean live litter size on day 4, g  13.50 + 3.60  14.00 + 2.00  13.70 + 2.21
 Cumulative loss on day 4, %  6.51 + 8.11  5.43 + 8.98  6.85 + 7.02
 Litter affected, N  5  5  7
       
 Total litter size on day 4 p.p., N  135  140  137
 Mean litter weight day 4 p.p., g  113.3 + 24.40  119.4 + 20.51  102.4 + 13.80
 Mean pup weight day 4 p.p., g  8.55 + 1.15  8.56 + 0.94  7.57 + 1.04

 Sex ratio:

 

 

 

 Sex ratio at birth, % males

 52.30 + 20.76

 49.82 + 12.47

 53.39 + 18.85

 Sex ratio on day 4 p.p., % males

 52.45 + 20.11

 50.25 + 12.88

 54.85 + 19.41

Applicant's summary and conclusion

Conclusions:
On the basis of the above results, treatment-related effects on pregnancy and on early lactation of the offspring, were observed in dams dosed at 80 mg/kg/day. Some treatment-related effects (as minor clinical signs, reductions in body weight) were also noted during the mating and gestation periods in the high dose males and females.
Reproductive parameters such as fertility index, pre-coital interval and copulatory index were not affected by treatment in neither sex at any dosage. The gestation length was not altered.

Adverse effects were seen in the high dose females (reductions in the number of implantation sites and of corpora lutea) and at parturition and total litter loss by cannibalism by day 1. Since these effects in the high dose females were observed in the presence of a marked maternal toxicity (severe reductions in body weight and body weight gain, severe changes in the liver and thymus) a correct evaluation/interpretation of the reproductive effects could not be done at this dose level.

No adverse effects on reproduction and pup development were observed in animals dosed at 20 mg/kg/day or 5 mg/kg/day. However, minimal to moderate effects on liver were observed at microscopic examination in some animals dosed at 20 mg/kg/day (parental males).
The NOAEL was set at 20 mg/kg/day for reproductive parameters and development.
Executive summary:

The purpose of this study was to investigate the effects of the test substance on reproduction, in 10 male and 10 female rats and on the development of offspring, when treated for at least 2 weeks before mating and throughout the gestation and lactation periods, until Day 3 post partum. Males received doses of 0, 5, 20 and 60 mg/kg/day, and females received doses of 0, 5, 20, and 80 mg/kg/day.


The potential liver toxicity was also evaluated at a lower dose of 0.5 mg/kg/day in a satellite group of 5 males and 5 females.


The study design was based on the procedures described in OECD guideline number 421, “Reproduction/ Developmental Toxicity Screening Test” (1995) and performed in compliance with GLP.


 


Minor clinical signs (piloerection, occasional rales) were detected in the high dose males and females from the third week in vivo phase (mating phase). Reductions in body weight/body weight gain and food consumption were observed in males and females from this dose group starting from the mating phase. Statistically significant reductions of body weight were still present in the high dose females during gestation period and in the males up to termination. Some effects (minor clinical signs, reductions in body weight/ body weight gain) were also noted during the mating period in the high dose males and females and during the gestation period in the high dose females.


At macroscopic and microscopic examination, treatment-related findings were found in the liver, coagulating glands, seminal vesicles, prostate, ovaries and in the thymus of the high dose animals. No treatment-related changes were seen in the testes. Reproductive parameters such as fertility index, pre-coital interval and copulatory index were not affected by treatment in neither sex at any dosage.


Adverse effects seen in the high dose females consisted of reductions in the number of implantation sites and of corpora lutea. At parturition, litters were lost by cannibalism or were smaller in size and weight, before total loss by day 1. Reductions in mean total and individual litter weights (when calculable) were the main treatment-related effects observed in dams dosed at 80 mg/kg/day in this study.


Since these effects in the high dose females were observed in the presence of a marked maternal toxicity (severe reductions in body weight and body weight gain, severe changes in the liver and thymus) a correct evaluation/interpretation of the reproductive effects could not be done at this dose level.


No significant effects were observed in animals dosed at 20 mg/kg/day. The slight reduction in mean pup weight seen on Day 1 was not considered to be significant as the mean total litter weight was not significantly reduced and, in the majority of cases, parturition occurred on Day 21 (not on Day 22 as in the controls). No effects on fertility, pregnancy and early lactation of the offspring were observed in animals dosed at 5 mg/kg/day. At macroscopic and microscopic examination no treatment-related lesions were seen in animals treated at 5 and 20 mg/kg/day. However, effects on liver were observed at microscopic examination in some parental animals dosed at 20 mg/kg/day. No treatment related changes were seen at the histopathological examination of the liver performed in satellite group animals, dosed at dosed at 0.5 mg/kg/day for 28 consecutive days.


Based on the results of this study, the NOAEL for reproductive toxicity and development is set at 20 mg/kg/day.