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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Remarks:
screening study
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:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
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
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
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
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.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- 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
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

Examinations

Maternal examinations:
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were treated to visualize possible hemorrhagic areas of implantation sites.

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, if necessary.

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)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

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.
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.
Indices:
Litter size (birth index), post-natal loss (PND0-4) (viability index).

[Also gestation time, gestation index, precoital time, conception index, corpora lutea count, implantation rate, post implantation loss.]

Historical control data:
provided and relevant to comment on organ weight changes (below)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
ORGAN WEIGHTS
In females at 500 mg/kg bw/day, 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 at all doses. This finding in the urinary bladder was said mainly to correlate with transitional cell hyperplasia observed at microscopic level.
HISTOPATHOLOGY: NON-NEOPLASTIC
Kidneys: At 500 mg/kg bw/day, multifocal tubular degeneration/regeneration was noted in all males and 2 females, and in 1 male this was associated with minimal hyaline casts. Tubular simple dilation was noted in 4 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 3 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 6/6 males and 5/5 females and correlated the hyperplastic findings in urinary bladders. Renal pelvic dilation was also observed in the 100 mg/kg bw dose group (4/5 females).
Urinary Bladder: Perivascular lymphoid cell infiltration was noted in 10/10 males and 6/7 females at 100 mg/kg bw/day, in 10/10 males and 7/8 females at 250 mg/kg bw/day and in 7/10 males and 10/10 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 8/10 males at 100 and 250 mg/kg bw/day, and 10/10 males at 250 mg/kg bw/day. In females, dilation in 3/7 at 100 mg/kg bw/day, 4/8 at 250 mg/kg bw/day, and 10/10 at 500 mg/kg bw/day was observed. Minimal to moderate bladder congestion occurred in all males at all dose levels. In females, congestion was observed in 6/7 at 100 mg/kg bw/day, 7/8 at 250 mg/kg bw/day, and 10/10 at 500 mg/kg bw/day. In each one male and one female at 250 mg/kg bw/day it was associated with slight hemorrhage.
Jejunum: At 500 mg/kg bw/day, multifocal lymphangiectasis of villi was noted in all males and females and in 2 males and 1 female at 250 mg/kg bw/day.
Liver and Thyroid: At 500 mg/kg bw/day, the liver cell hypertrophy noted in 2/5 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 (maternal animals)

Dose descriptor:
LOAEL
Remarks:
maternal toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Basis for effect level:
other: effects to urinary bladder at lowest dose tested

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: urinary system

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
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 (fetuses)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental 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 developmental 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.