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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:
14 Jan - 20 Oct 2009
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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Lot/batch No.: 77997MJ
- Expiration date of the lot/batch: 2014-01-13

Test animals

Species:
rat
Strain:
other: Han Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Labs Ltd., Wölferstrasse 4, 4414 Fuellinsdorf, SWITZERLAND
- Age at study initiation: 11 wk
- Weight at study initiation: 294-330 g (m), 178-213 g (f)
- Housing: 1/Makrolon type 3 cage
- Diet: standard diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 20-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2009-01-14 To: 2009-04-06

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Prepared weekly. Homogeneous suspension maintained with magnetic stirrer during dosing.
VEHICLE
Dried deacidified corn oil
- Justification for use and choice of vehicle (if other than water): none given
- Concentration in vehicle:
- Amount of vehicle (if gavage): dose volume 5 ml/kg bw
- Lot/batch no.: 37899577
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 wk
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC-FID. Stability and homogeneity verified at start of study and during 2nd last week of dosing.
Duration of treatment / exposure:
toxicity males: from 2 weeks prior to mating for at least 4 wk
toxicity/reproductive females: from 2 weeks prior to mating for at about 7 wk

Frequency of treatment:
daily, 7/days/wk
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: prior range finding study (Harlan B88762)
- Rationale for animal assignment (if not random): random with consideration for body weight
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just prior to sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (18 h)
- How many animals: 5

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just prior to sacrifice
- Animals fasted: Yes (18 h)
- How many animals: 5

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION/FOB: Yes
- Time schedule for examinations: 1 or 2 days prior to sacrifice (males) or postpartum day 3/4 (females)
- Dose groups that were examined: 5/sex, all groups
Cage-side observations: unusual body movements (e.g. tremors, convulsions), abnormal behavior (e.g. circling, stereotypy) and posture as well as resistance to removal.
Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.
Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.
Categorical observations (can be made any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or feces, soiling, general abnormalities, posture.
Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
Locomotor activity (low beam counts)

Oestrous cyclicity (parental animals):
not recorded
Sperm parameters (parental animals):
not recorded
Litter observations:
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 postpartum.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals day 28/29
- Maternal animals:PND 4/5


GROSS NECROPSY
All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred. For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.


HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues indicated in OECD 422 were prepared for microscopic examination and weighed.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, ifnecessary.

Postmortem examinations (offspring):
All pups were examined macroscopically for any structural changes
Statistics:
Means and standard deviations of various data were calculated. The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex. The Steel-test (many-one rank test) was applied instead of the Dunnett test when the data could not be assumed to follow a normal distribution. Fisher's exact-test was applied to breeding data and the macroscopical findings.
Reproductive indices:
Gestation time, precoital time, corpora lutea count, implantation rate, post implantation loss.
Percentage mating = (Females mated / Females paired) * 100
Fertility index = (Females achieving a pregnancy / Females paired) * 100
Conception rate = (Females achieving a pregnancy / Females mated) * 100
Gestation index = (Number of females with living pups / Number of females pregnant) * 100
Offspring viability indices:
Litter size: Birth index = (number of pups born alive / number of implantations) * 100
Post-natal loss (PND 0-4): Viability index = (number of alive pups on day 4 p.p. / number of pups born alive) * 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
500 mg/kg bw: reduced body weight and body weight gain
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
500 mg/kg bw: reduced body weight and body weight gain
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
500 mg/kg bw: increased kidney weights (male)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
100, 250, 500 mg/kg bw: thickened urinary bladder
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
100, 250, 500 mg/kg bw: hyperplasia, dilatation in the urinary bladder
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

Details on results (P0)

Mean precoital time, fertility and gestation indices and conception rate were not affected by the treatment with the test item.
Implantation rate and post-implantation loss were also not affected by the treatment with the test item.

CLINICAL SIGNS AND MORTALITY
No treatment-related effects.

BODY WEIGHT AND WEIGHT GAIN
In males: at 500 mg/kg bw/day mean body weight gain was statistically significantly reduced during the pre-pairing period. This resulted in a statistically significantly decrease in body weight throughout the whole study. In females: at 500 mg/kg bw/day mean body weight gain was statistically significantly reduced between days 8 and 14 of the gestation resulting in a decrease of mean body weight between days 11 and 20 of gestation.

FOOD CONSUMPTION
500 mg/kg bw: reduced food consumption in the pre-pairing period (male/female), increased food consumption in the after-pairing period (male)
HAEMATOLOGY
No treatment-related effects.

CLINICAL CHEMISTRY
In males, at 250 and 500 mg/kg bw/day, the concentration of urea was statistically significantly increased in dose-dependent manner. The concentration of bile acids was statistically significantly increased but without showing a dose-dependent pattern. At 500 mg/kg bw/day, the concentration of cholesterol was also statistically significantly increased.

NEUROBEHAVIOUR/FOB
No treatment-related effects.

ORGAN WEIGHTS
The report highlights that at 500 mg/kg bw/day, absolute and relative weight of the kidneys was statistically significantly increased in males. In addition, in females at this dose, thymus weight was reduced (absolute, relative to body and relative to brain), although these values were within the range of historical controls. Female liver and kidney weights (relative to body) were also increased at the top dose; again within the range of historical controls. [The report incorrectly notes increased thymus and reduced liver weights for this group.]

GROSS PATHOLOGY
The urinary bladder was thickened in all treated groups (100 mg/kg bw/day 6/10 m, 2/10 f; 250 mg/kg bw/day 7/10 m, 6/10 f; 500 mg/kg bw/day 9/10 m, 7/10 f). This finding in the urinary bladder was said mainly to correlate with transitional cell hyperplasia observed at microscopic level.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopically, the test item-related lesions recorded were:
Kidneys: At 500 mg/kg bw/day, multifocal tubular degeneration/regeneration was noted in all males and two females, and in one male this was associated with minimal hyaline casts. Tubular simple dilation was noted in four males and all females. Increased incidence of focal tubular degeneration/regeneration was observed in females. Transitional cell hyperplasia was noted in all males and females. At 250 mg/kg bw/day, multifocal tubular degeneration/regeneration was observed in three males. Transitional cell hyperplasia was noted in all males and females. The hyperplastic lesions were accompanied by an increased incidence of renal pelvic dilation in six/six males and five/five females and correlated the hyperplastic findings in urinary bladders.
Urinary Bladder: Perivascular lymphoid cell infiltration was noted in ten/ten males and six/seven females at 100 mg/kg bw/day, in ten/ten males and seven/eight females at 250 mg/kg bw/day and in seven/ten males and ten/ten females at 500 mg/kg bw/day. At all dose levels, minimal to moderate transitional cell hyperplasia was observed in all males and females. These hyperplastic lesions were accompanied by minimal to slight dilation in each eight/ten males at 100 and 250 mg/kg bw/day, and ten/ten males at 250 mg/kg bw/day. In females, dilation in three/seven at 100 mg/kg bw/day, four/eight at 250 mg/kg bw/day, and ten/ten at 500 mg/kg bw/day. Minimal to moderate bladder congestion in all males at all dose levels. In females, six/seven at 100 mg/kg bw/day, seven/eight at 250 mg/kg bw/day, and ten/ten at 500 mg/kg bw/day, in each one male and one female at 250 mg/kg bw/day associated with slight hemorrhage.
Jejunum: At 500 mg/kg bw/day, multifocal lymphangiectasis of villi was noted in all males and females and in two males and one female at 250 mg/kg bw/day.
Liver and Thyroid: At 500 mg/kg bw/day, the liver cell hypertrophy noted in two/five females and consequent increase of follicular cell hypertrophy in the thyroid gland was considered to be an adaptive effect and therefore, not adverse.

Effect levels (P0)

Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: urinary bladder effects at lowest dose tested

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

The mean number of pups at first litter check was not affected by the treatment with the test item. The sex ratio was also not affected. No abnormal pups were noted at any dose level. During the lactation period, pup weight gain at 500 mg/kg bw/day was reduced compared to the controls, but did not attain statistical significance. The reaction was due primarily to lower weight gain which occurred in two litters. Excluding the lower pup weight which occurred in these two litters at 500 mg/kg bw/day, mean pup weight gain was not considered to be affected by the treatment with the test item.

At necropsy of the pups, the incidence of pups with no milk in the stomach did not give any indication of a test item-related effect.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No adverse reproductive effects at highest dose tested

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
A well reported oral combined repeated dose/reproductive and developmental screening study, conducted according to the current guideline and in accordance with GLP, reported a NOAEL for reproductive effects at the highest tested dose of 500 mg/kg bw/day. General systemic parental effects were reported at the lowest tested dose of 100 mg/kg bw/day.