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Toxicity to reproduction

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Administrative data

Endpoint:
reproductive toxicity, other
Remarks:
other: Reproductive function and early embryonic development.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-March 2002 to 04-Nov-2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Guidelines for Reproductive/Developmental Toxicity Studies on Drugs (Notification No. 316 of the Pharmaceutical Affairs Bureau, Japanese Ministry of Health and Welfare, April 14, 1997)
Qualifier:
according to guideline
Guideline:
other: Guidelines for Reproductive and Developmental Studies of Drugs (Revised Version) (Notification No. 1834 of the Pharmaceutical Judges Bureau, Japanese Ministry of Health and Welfare, December 27, 2000).
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): RM-60
- Physical state: yellow powder
- Analytical purity: >98%
- Purity test date: 14-March-2002 and 10-June-2002
- Lot/batch No.: 010755D1AA
- Stability: stable for at least 1 week under refrigerated conditions and at least 1 day at room temperature.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc. (Hino Breeding Center)
- Age at study initiation: males (8 weeks), females (7 weeks)
- Weight at study initiation: males (267 to 306 g), females (170 to 209 g)
- Fasting period before study: none specified
- Housing: stainless steel cages (same sex during quaranteen and acclimitization; individually during initial dosing; one-to-one for mating)
- Diet (e.g. ad libitum): CRF-1 Oriental Yeast Co, ad libitum
- Water (e.g. ad libitum): tap, ad libitum
- Acclimation period: 14 days following a 5 day quaranteen.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 41 - 61
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES (start of administration of test substance to necropsy) : From: 8 April 2002 To: 6-10 May 2002 (females) and 28-31 May 2002 (males)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: RM-60 was measured out for each concentration and pulverized well using a mortal and pestle. Then PEG 400 was added to RM-60 little by little to make suspensions at specified concentrations. Dosing preparations were made up at least once a week, divided into daily doses, and stored in a storage container placed in the test article storage room of the testing facility under refrigerated, light-shielded conditions until use.

VEHICLE
- Concentration in vehicle: 1%, 3%, 10%
- Amount of vehicle (if gavage): 10 ml/kg-bw
Details on mating procedure:
- M/F ratio per cage: one-to-one
- Length of cohabitation: all pairs mated within 5 days.
- Proof of pregnancy: positive vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations and homogeneity of the test article in the dosing preparations were determined by HPLC prior to and after the dosing period. The concentrations were between 100.9 and 104.0% (acceptable range: 90 - 110%), and the coefficient of variance (C.V.) indicating homogeneity was between 0.3 and 2.1% (acceptable values: 5% or lower). Since the values were within the acceptable ranges, there were no problems with the concentrations or homogeneity of the test article. Dosing preparations remaining after administration were discarded.
Duration of treatment / exposure:
Males: until the day before the mating period (Day 14 of adrninistration; the first dosing day was defined as Day 1
of administration), throughout the mating period (for 28 days), and for 8 - 11 days thereafter; total of 50 - 53 days.
Females: until the day before the mating period (Day 14 of administration), throughout the mating period (for 5 days at the longest),
and for 7 days after copulation (until Day 7 of pregnancy); total of 23 - 27 days.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
No effects were noted at 1000 mg/kg either in a repeated dose toxicity study of RM-60 by 2-week oral administration in rats or in a study of RM-60 on embryo-fetal development in rats. Accordingly, the high dosage level in the present study was set, as the limit dose prescribed in the Guidelines for Toxicity Studies of Drugs, at 1000 mg/kg/day. The intermediate and low dosage levels were set at 300 and 100 mg/kg, respectively, by decreasing geometrically by a factor of about 3.
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, once before and once after administration of dose.

BODY WEIGHT: Yes
- Time schedule for examinations: Two times per week.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, one time per week.

Oestrous cyclicity (parental animals):
Once per day from the first dosing day to the day when copulation was confirmed.
Sperm parameters (parental animals):
Parameters examined in all P male parental generations:
nr. of sperm, sperm motility (motile sperm rate (%), progressive sperm rate (%), path velocity, straight line velocity, curvilinear velocity, amplitude of lateral head displacement, beat cross frequency), survivability, morphology

For sperm motility, survivability, and morphology: Right cauda epididymis was cut into strips in culture fluid for sperm warmed to 37°C and left standing for about 5 minutes.
For the nr. of sperm: Sperm suspensions were prepared by the left cauda epididymis.

Equations used:
- No. of sperms/g of the left cauda epididymis = No. of sperms/caudal weight of left epididymis (g).
- Sperm viability rate (%) = [(No. of surviving sperms + No. of sperms which died in the course of sperm analysis) / No. of sperms observed] x 100.
- Sperm survivability rate (%) = (No. of surviving sperms / No. of sperms observed) x 100.
- Abnormal sperm rate (%)= (No. of abnormal sperms / No. of sperms observed) x 100.
Litter observations:
Mortality of embryos and implantation sites were observed for each dam macroscopically. After being observed, the embryos were fixed and preserved in 10% neutral buffered formalin together with the uterus and placenta removed from respective dams.
Postmortem examinations (parental animals):
The males were sacrificed the day after the last dosing day by bleeding from the abdominal aorta under ether anesthesia and necropsied.
The testes and epididymides were removed, and these organs and the caudae epididymidis were weighed. The relative testis and epididymis weights were calculated by dividing the respective absolute organ weight by the final body weight.
The removed bilateral testes and bilateral capita epididymidis were fixed in Bouin's solution for 2 - 3 hours and preserved in 10% neutral buffered formalin. The left cauda epididymis was stored frozen (set at -80°C) until the day of examination.

Females which had copulated were sacrificed on Day 13 of pregnancy (13 days after copulation) by bleeding from the abdominal aorta under ether anesthesia and necropsied for pregnancy.
Regarding the females whose pregnancy was confirmed, the number of corpora lutes and number of implantation sites were counted, and the ovaries and uterus (with embryos and placenta) were fixed and preserved in 10% neutral buffered formalin.
Regarding the females in which implantation was not found, the ovaries and uterus were removed. The uterus was stained with 10% ammonium sulfide, and the absence of implantation scars was re-confirmed. Then the uterus was fixed and preserved in 10% neutral buffered formalin together with the ovaries.
Statistics:
Data were analyzed according to the statistical methods described as follows.
Significance tests were performed between the control group and each of the groups treated with RM-60. Probabilities less than 5% (1% for Bartlett's test) were considered statistically significant. Data for non-pregnant females obtained after copulation were excluded from the totals.
Group mean values and standard deviations were calculated for body weight (with body weight gain), food consumption, organ weights (relative organ weight included), number of sperms, number of sperms/g of the left cauda epididymis, sperm motility, sperm survivability, abnormal sperm rate, number of estrous cases (until the day before pairing), days till copulation after pairing, number of corpora lutea, number of implantation sites, implantation rate, number of pre-implantation losses, pre-implantation loss rate, number of dead embryos (number of post-implantation losses), dead embryo rate (post-implantation loss rate), and number of live embryos.
Subsequently, the data were tested by Bartlett's method for homogeneity of variance. When the variances were homogenous, Dunnett's method was used. When the variances were heterogeneous, a Dunnett-type method using a rank order was used.
The fertility index was analyzed by chi square tests.
Reproductive indices:
Copulation index (%) = (No. of animals which copulated / No. of animals which were paired) x 100.
Days till copulation after pairing = day of confirmation of copulation - first day of the mating period.
Fertility index (%) = (No. of pregnant females / No. of females which copulated) x 100.
Pre-implantation loss rate (%) = [(No. of corpora lutea - No. of implantation sites) / No. of corpora lutea] x.100.
Implantation rate (%) = (No. of implantation sites / No. of corpora lutea) x 100.
Dead embryo rate (post-implantation loss rate) (%) = (No. of dead embryos / No. of implantation sites) x 100.
Offspring viability indices:
Not applicable.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Loose stool and transient salivation, but also noted in control group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
similar in test substance treated groups to those in the control group, and no significant differences were seen on any day of measurement. Also, no significant differences from the control group were seen in body weight gain.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
similar in test substance treated groups to those in the control group, and no significant differences were seen on any day of measurement.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No abnormalities were noted in the testes, capita epididymidis, or ovaries examined from the animals which showed no fertility (fertilizability).
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No significant differences were seen in the number of estrous cases during 14 days before the dosing period or during 14 days after the dosing period between any of the treated and the control groups.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No significant differences were seen in the motile sperm rate, progressive sperm rate, path velocity, straight line velocity, curvilinear velocity, amplitude of lateral head displacement, beat cross frequency, viability rate, survivability rate, abnormal sperm rate, or number of sperm of the left cauda epididymis between any of the treated and the control groups.
Reproductive performance:
no effects observed
Description (incidence and severity):
All pairs in each group copulated by the first mating. Only 1 pair each in the control and mid tose group had no fertility (fertilizability).
No significant differences were seen in the fertility index between any of the treated and the control groups.

Effect levels (P0)

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

Results: F1 generation

Details on results (F1)

Compared with the control group, the number of dead embryos (number of post-implantation losses) and dead embryo rate (post-implantation loss rate) were significantly higher in the low dose group, but these findings were not dose-dependent changes.
No significant differences were seen in the number of corpora lutea, number of implantation sites, implantation rate, number of pre-implantation losses, pre-implantation loss rate, or number of live embryos between any of the treated and the control groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no test substance related adverse effects observed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
It is concluded that the no observed adverse effect level of RM-60 is >=1000 mg/kg for general toxicity in dams, for reproductive functions of parent animals, and for early embryonic development.
Executive summary:

RM-60 at dosage levels of 100, 300, and 1000 mg/kg was administered orally by gavage to male rats daily before mating and throughout the mating period and to female rats daily before mating and throughout the mating period up to the early stages of pregnancy, and the effects of RM-60 on reproductive functions of male and female animals and early embryonic development were assessed. Regarding the parent animals, loose stool and salivation were noted in both sexes in the groups treated with RM-60, but these findings were also noted similarly in the control group. It is concluded, therefore, that these findings were not attributable to RM-60 but caused by PEG 400, the vehicle. No effects of RM-60 were noted in body weight, food consumption, or necropsy findings in either sex. Also, no effects of RM-60 were noted in male organ weights or sperm analysis parameters. In the examination for reproductive functions, no effects of RM-60 were noted in the number of estrous cases, days till copulation after pairing, copulation index, or fertility index. In the observation of embryos, no effects of RM-60 were noted in the number of corpora lutea, number of implantation sites, implantation rate, number of pre-implantation losses, pre-implantation loss rate, number of dead embryos (number of post-implantation losses), dead embryo rate (post-implantation loss rate) or number of live embryos.

From the above results, it is concluded that the no observed adverse effect level of RM-60 is >=1000 mg/kg for general toxicity in dams, for reproductive functions of parent animals, and for early embryonic development.

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