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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental toxicity screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-01-14 to 2009-10-20
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
Remarks:
no significant deviation
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

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 Fűllinsdorf, 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 days

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 oral 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
- 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:
CG-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:
toxicity males: 10
toxicity/reproductive females: 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

Observations and examinations performed and frequency:
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: just prior to sacrifice
- 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.



Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (range of tissues as specified in OECD 422)
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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
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.

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 eacheight/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.

HISTORICAL CONTROL DATA (if applicable)
See comment on organ weights above.

OTHER
Measured doses were in the range 94-103% of nominal.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Sex:
male/female
Basis for effect level:
other: effects in urinary bladder at all doses (>=100 mg/kg bw/day)
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: effects on urinary bladder

Target system / organ toxicity

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

Any other information on results incl. tables

Increased urea and cholesterol can both be indicative for kidney effects.

At all dose levels (100, 250 and 500 mg/kg bw/day) during the histopathology examination, perivascular lymphoid cell infiltration and transitional cell hyperplasia of the urinary bladder were observed in males and females.Therefore, based on the findings in urinary bladder noted in all test item-treated groups, a general NOAEL could not be established.

Applicant's summary and conclusion

Conclusions:
A well reported oral combined repeated dose/reproductive and developmental screening study, conducted according to OECD 422 and in accordance with GLP, did not identify a NOAEL for the registered substance; effects to the urinary bladder were reported at the lowest tested dose of 100 mg/kg bw/day.