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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-06-02 - 2008-10-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Storage Conditions: ambient temperature, in the dark

Test animals

Species:
rat
Strain:
other: Crl: CD®(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent on the 28 May 2008 and the 22 July 2008, respectively to provide 96 animals of each sex for the study
- Age at study initiation: 4 weeks
- Weight at study initiation: (P) Males:81-99g; Females: 150-173g;
- Housing: The animals were initially housed two per cage. The cages were made of polypropylene, with solid bottoms and stainless steel mesh tops, and measured ca 48 x 37.5 x 21 cm. A stainless steel food hopper and polypropylene or a polycarbonate water bottle was provided for each cage. The cages were suspended on a series of racks. Male and female cages were racked separately.
A few days prior to mating, males were transferred into individual grid-bottomed cages (ca 59 x 38.5 x 20 cm) of a similar design. Excreta were collected on a tray lined with absorbent paper suspended beneath the cage. After mating, the males were housed 2 per cage with their original cage-mates in solid bottomed cages. Mated females were transferred into individual ca 48 x 37.5 x 21 cm solid-bottomed cages. Just prior to the anticipated parturition, some white paper tissue was provided as nesting material.
On Day 101 of treatment (Day 0 = First day of treatment); Animals 63 and 64 (Group 3M) were suspected of fighting and were removed from each other and housed singly; these animals were re-housed together on Day 107 of treatment.
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded) SQC, supplied by Special Diets Services Ltd., Stepfield, Witham, Essex, UK was available to the animals ad libitum. The diet was supplied with a batch analysis for nutritive constituents and a range of significant contaminants. The analytical certificate for a batch used during the study has been retained in the study archive.
- Water (e.g. ad libitum): The animals had access to domestic quality mains water ad libitum. The supply is analyzed regularly for dissolved and suspended materials, including a range of significant contaminants. The analytical certificate of a typical recent analysis has been retained in the study archive.
- Acclimation period: The animals were acclimatized in the Charles River animal room for a minimum of 12 days from arrival until commencement of treatment. All the animals were clinically examined on arrival for signs of abnormality or disease. No such signs were found and the animals were accepted for use on this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): Light hours were 0700-1900 hours.

IN-LIFE DATES:
Experimental Start Date: 02 June 2008
Experimental Completion Date: 13 October 2008

- Environmental Enrichment
To provide environmental enrichment, wooden chewsticks were made available to the animals, as appropriate. The chewsticks were not considered to contain any additional substances in sufficient concentrations to influence the outcome of the study.

- Animal Identification
Each animal received a subcutaneously implanted electronic identification chip identifying it individually within the study and which corresponded to that animal’s number. It was also given a cage card which was color coded for treatment group and marked with the study number, animal number, cage number and treatment group.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared at approximately weekly intervals and used within the established stability period of 8 days (established in Charles River Laboratories Study No. 424799, Method 2479). An appropriate amount of test item was weighed and placed in a suitably sized glass container, and then the appropriate amount of vehicle was added and mixed by magnetic stirring until a visibly clear, homogenous suspension was obtained. The vehicle used was 2% medium viscosity CMC containing 0.2% Tween 80. Full details of the vehicle used are retained in the study raw data. To correct for purity, a correction factor of 1.04 was used for the calculation of doses on this study.

The animals were dosed once daily by oral gavage, at a dose volume of 10 mL per kg body weight, using a plastic gavage. The volume to be administered to each animal was determined by the weight of the animal as measured at the time of administration, except during the fetal period of gestation (from day of 16 gestation until parturition was complete); throughout this period the dose volume was determined by the weight of the animal on Day 16 of gestation. The animals were dosed once daily, at approximately the same time each day. The F0 males were dosed for 10 weeks prior to mating. The F0 females were dosed for two weeks prior to mating. Dosing for both the sexes continued throughout mating and throughout gestation and lactation for the females.
Details on mating procedure:
- M/F ratio per cage: Animals were paired in numerical order on a 1 male to 1 female basis
- Length of cohabitation: Each female was transferred to the cage of its appropriate co-group male near the end of the working day, where it remained until mating had occurred. Each female was left with its designated male for a maximum of 14 nights.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- The time taken to show a positive mating sign and the number of failed opportunities to mate (estrous passed without sign of mating) was evaluated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the formulations was undertaken with regard to concentration and homogeneity from formulations prepared for use on Day 1 dosing of males, Day 1 dosing of females and from Week 11 of dosing. On each occasion, ca 1mL triplicate samples were taken (top, middle and bottom) from the formulations for groups 1-3 and ca 0.5mL triplicate samples from the group 4 formulations. These were analyzed to provide data on concentration and homogeneity. The samples were assayed using methodology established under Charles River Study No. 424799, Method No.2479.
The results of the analysis of the dosing formulations were within ±9.5% of the nominal concentration indicating acceptable accuracy of formulation. The low coefficients of variation (<4%) indicated that the formulations were homogenous.
Duration of treatment / exposure:
Test item was administered for 10 weeks prior to mating for males and two weeks prior to mating for the females, then throughout mating, gestation and lactation until termination after the litter had reached Day 21 of lactation.
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
400 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
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were agreed with the Sponsor after evaluation of existing relevant toxicological data, including a developmental toxicity study in rats (Charles River Study No. 494929).
- Rationale for animal assignment:
Animals were allocated to treatment groups by the use of randomly sequenced numbers, in such a way that each full rack contained similar numbers of representatives from all groups.
- Other:
The spare animals were numbered 193-194 (males) and 195-196 (females). Animal 116 was killed prematurely on Day 4 of dosing. The signs for this animal were consistent with damage caused by the dosing procedure and therefore it was considered appropriate to replace this animal with Animal 195 on Day 4 of treatment of the females.

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
All the animals were examined during each day, from commencement of dosing. The onset, intensity and duration of any clinical signs were recorded. Once each week (starting during the pre-trial period) all adult animals were given a detailed clinical examination, including appearance, movement and behavior patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
For both sexes, body weights of F0 animals were recorded one week prior to the first day of dosing, then daily throughout the dosing period until termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Males: The weight of the food consumed was recorded weekly from one week before commencement of treatment until placement of males in individual cages prior to mating. Consumption over complete weeks was also recorded after the 14 day mating period.
- Females: The weight of the food consumed was recorded weekly from one week before the commencement of treatment until the placement of males in individual cages prior to mating. Consumption was also recorded over the periods Days 0-7, 7-14 and 14-20 of gestation, and Days 0-7 and 7-14 of lactation.

OTHER:
Any deficiencies in maternal care were recorded. Points looked for included inadequate construction and cleaning of the nest, pups left scattered and cold, physical abuse of pups, apparent inadequate lactation or feeding.
Oestrous cyclicity (parental animals):
Vaginal lavages were taken early each morning, commencing on the day of pairing, until mating had occurred. The stage of oestrous observed in each vaginal lavage was also recorded.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
- Testes (weighed individually), fixed in Bouin’s fluid
- Epididymides (weighed individually)
- Seminal Vesicles and coagulating gland
- Prostate gland
Litter observations:
The number of live pups born and the number found dead in each litter were recorded as soon as possible after completion of parturition. The live pups were sexed, counted, examined for the presence of milk in the stomach and for any externally visible abnormalities daily up to Day 4 of lactation and then on Days 7, 14 and 21. The pups were separated by sex and weighed en masse on Days 1, 4, 7 and 14 of lactation. On Day 21 the pups were weighed individually. When possible, any pups that were found dead or killed during lactation were sexed and appropriately examined. Prior to Day 14 of lactation, externally abnormal decedent pups were preserved; externally normal ones were discarded. On or after Day 14 of lactation, decedent pups were necropsied.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals. Termination of the F0 males was at approximately the same time as the F0 females.
- Maternal animals: All surviving animals. Termination of the F0 females was at or shortly after weaning of their litters (Day 21 of lactation).

GROSS NECROPSY
Necropsy consisted of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. Any gross lesions were described in terms of location, size, shape, color, consistency, number and any other relevant characteristics. Representative samples of abnormal tissues were taken and fixed in neutral buffered 10% formalin. The following organs were also fixed:

Ovaries
Uterus (including cervix) and vagina
Testes (weighed individually), fixed in Bouin’s fluid
Epididymides (weighed individually)
Seminal Vesicles and coagulating gland
Prostate gland
Pituitary gland

The reproductive tract of all females was examined for signs of implantation with the number of implantation sites being recorded.
Postmortem examinations (offspring):
SACRIFICE
At terminal necropsy, 2 male and 2 female pups (where they were available) were necropsied.

GROSS NECROPSY
- Gross necropsy consisted of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Representative samples of any grossly abnormal tissues were persevered in 10% formalin. The remaining pups in each litter were checked for externally visible abnormalities at the time of killing. Any abnormal pups found were necropsied as previously described.

Offspring killed or found dead before Day 14 of lactation were checked for the presence of milk in the stomach and the presence of externally visible abnormalities. Any abnormal pups were preserved in 10% formalin or methylated ethyl alcohol as appropriate for possible future examination. Externally normal decedents were discarded.

Offspring killed or found dead on or after Day 14 of lactation were necropsied. This consisted of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of grossly abnormal tissues were preserved in 10% formalin and the carcasses were then discarded.
Statistics:
Body weights and food consumption (for males throughout the study and for females prior to mating) and organ weigh data were analyzed for homogeneity of variance using the “F-Max” test. If the group variances appeared homogenous, a parametric analysis of variance (ANOVA) was used and individual between group comparisons were made using Fisher’s F-protected LSD method via Student’s t-test. If the variances were heterogeneous, log or square root transformations were performed in order to stabilize the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and a pairwise comparison was made using chi squared protection (via z tests, the nonparametric equivalent of Student’s t-test).
In addition to the ANOVA, organ weights were analyzed by analysis of covariance (ANCOVA) using terminal body weight as covariate. For other parameters, no formal statistical analyses were considered necessary; interpretation of the data being by inspection of the individual and group values.
Reproductive indices:
Fertility Index (male) = Number siring a litter / Number Paired
Fertility Index (female) = Number Pregnant / Number Paired
Gestation Index = Number bearing live pups / Number pregnant
Birth Index = Total number of pups born (live and dead) / Number of implantation scars
Live Birth Index = Number of pups live on Day 0 of lactation / Total number born (live and dead)
Offspring viability indices:
Viability Index = Number of pups live on Day 4 of lactation / Number live on Day 0
Lactation Index = Number of pups live on Day 21 of lactation / Number live on Day 4
Overall Survival Index = Number of pups live on Day 21 of lactation / Total number born (live and dead)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical observations that were considered to be related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Group mean body weight gains of males throughout the study were comparable with Control. Group mean body weight gains of females prior to mating and throughout gestation and lactation were similar to Control.
Description (incidence and severity):
Group mean food consumption of males throughout the study were comparable with Control. Group mean food consumption of females prior to mating and throughout gestation and lactation were similar to Control.
Food efficiency:
no effects observed
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:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance, fertility and duration of gestation were similar in all groups.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Slight intergroup differences in the number of implant sites and survival indices were too small to indicate any association with treatment. Mean litter and pup weights were essentially similar in all groups
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean litter and pup weights were essentially similar in all groups.
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:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Abnormalities Among Pups
The following abnormalities were observed among pups:
Litter 105 (Group 1): One female pup had a small swelling on ventral abdomen on Day 7.
Litter 126 (Group 2): One pup difficult to sex, suspect female, had no tail or anus evident. Killed on Day 1.
Litter 129 (Group 2): One male pup killed on Day 0 due to gasping, cold to touch, dark in color and unfed.
Litter 137 (Group 2): Days 1-21: One male and one female pup with short, threadlike tails.
Litter 149 (Group 3): Day 0, one female pup killed due to gasping, dark in color and unfed.
Litter 153 (Group 3): One female pup killed on Day 0 due to muzzle being damaged by dam.
Litter 164 (Group 3): Days 4-16 one male pup with swelling on ventral abdomen.
Litter 180 (Group 4): One male pup killed prematurely due to large head and reduced motor function on Day 19.
Litter 184 (Group 4): Day 0: One male pup killed due to gasping, cold to touch, unfed and pale in color. One female pup found dead with right hindlimb not evident. Right fore limb also found to be damaged at pathology external examination.

None of these abnormalities were considered to be attributable to treatment.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
At levels up to 1000 mg/kg/day, there were no obvious effects of treatment on clinical observations, necropsy findings, body weight gain or food consumption. Mating performance, fertility, duration of gestation, litter size and survival, and litter and pup weights did not indicate any obvious adverse effect of treatment at any of the dose levels tested. Under the conditions of this study, the no observed effect level (NOEL) for both parental and reproductive effects was 1000 mg/kg/day.
Executive summary:

In a One-Generation study according to OECD guideline 415 and in compliance with GLP, the test article was evaluated regarding its effects on general reproductive processes in rats. To that end, the test article was administered to males from 10 weeks prior to mating and to females from two weeks prior to mating, throughout mating, gestation and lactation until the litter had reached Day 21 of lactation. Sprague-Dawley rats were randomized into 3 test groups and 1 vehicle control group, each containing 24 males and 24 females. The animals were dosed once daily by oral gavage at dose levels of 0 (2% medium viscosity CMC containing 0.2% Tween 80), 150, 400, and 1000 mg/kg/day. The dose volume applied was 10 mL/kg body weight. Animals were regularly monitored for clinical signs of toxicity and for body weight and food consumption. The F0 females, along with their F1 pups, were killed shortly after weaning (day 21 of lactation). The males were killed at approximately the same time as the females. All adult (F0) animals were given a gross tissue examination along with 2 male and 2 female F1 pups from each litter. The reproductive organs from the adult (F0) animals were weighed (testes and epididymides only) and fixed. At levels up to 1000 mg/kg/day, there were no obvious effects of treatment on clinical observations, necropsy findings, body weight gain or food consumption. Mating performance, fertility, duration of gestation, litter size and survival, and litter and pup weights did not indicate any obvious adverse effect of treatment at any of the dose levels tested. Under the conditions of this study, the no observed effect level (NOEL) for both parental and reproductive effects was 1000 mg/kg/day.