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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
fertility, other
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (Hematology and clinical chemistry was performed only in males; Urinalysis not conducted)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990

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:
yes
Remarks:
Hematology and clinical chemistry was performed only in males; Urinalysis not conducted
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: about 10 weeks
- Weight at study initiation: 329.9 - 4045.8 g (males), 217.6 - 254.9 g (females)
- Housing: stainless steel cage for administration period, policabonate cage with wood chip during gestation and lactation period
- Diet: ad libitum (MF, Oriental Yeast Co., Ltd., Itabashi-ku, Japan)
- Water: ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 26
- Humidity (%): 53 - 61
- Air changes (per hr): 13 - 15 times/day
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% w/v sodium solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item was diluted in the vehicle once or twice a week. Diluted test item was stored in dark and cool place.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not described
- Amount of vehicle (if gavage): 2.5 mL/kg
- Lot/batch no. (if required): M7T4661
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: one night (or max. 2 weeks)
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): policabonate cage with wood chip
- Any other deviations from standard protocol: no deviation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Detail information was not available.
Duration of treatment / exposure:
Males, 49 days
Females, from 14 days before mating to day 3 of lactation
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
4, 20 and 100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the result of a preliminary study, maximum dose was set as 100 mg/kg bw /day

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (before and after administration)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39, 43, 46 and 49 for males; Day 1, 4, 8, 11 and 15 during pre-mating, Day 0, 4, 7, 10, 14, 17 and 21 during gestation, Day 0 and 4 during lactation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER:
Haematology and clinical chmistry
Oestrous cyclicity (parental animals):
Oestrous cycle length and normality were evaluated in females by vaginal smears prior to mating.
Sperm parameters (parental animals):
Parameters examined in all male parental generations: testis and epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals the day after last administration
- Maternal animals: All surviving animals at day 4 of lactation

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathology: Brain, heart, lungs, thyroids, thymus, liver, spleen, kidneys, adrenal, testis, epididymis, ovaries, bone marrow of femur,
Organweight: Brain, heart, lungs, thyroids, thymus, liver, spleen, kidneys, adrenal, testis, epididymis, ovaries,
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:
Bartlett test for body weight, food consumption, hematological result, myelogram, biochemistry result, duration of mating, count of estrus, estrous cycle, organ weight, gestational days, number of corpora lutea, number of implantations, number of litter, number of live newborns, body weight of live newbornes. In case of normal distribution, Dunnet multiple comparison was used to compare the control group. In case of not normal distribution, Steel multiple comparison was used. Chi-square test was used for percentage of succesful mating, percentage of the number of copulated, gestation inde and sex ratio of live newborns. Wilcoxon test was used for implant index, percentage of stillborns, birth index, sex ration of live newborns and viability index at day 4. Mann-Whitney U test was used for histopathological findings. For all test, p values < 0.05 was significant.
Reproductive indices:
- Copulation index: (number of pairs with successful copulations/number of pairs)x100
- Fertility index: (number of pregnant females/number of pairs with successful copulation)x100
- Implantat index: (number of implantation scars/number of corpora lutea)x100
- Gestation index: (number of females having live pups/number of pregnant dams)x100
Offspring viability indices:
- Sex ratio at birth: (number of male pups/number of female pups)x100
- Birth index: (number of live pups born/number of pups born)x100
- Stillbirth index: (number of stillborns/number of litters)x100
- Viability index: (number of live pups on Day 4 of lactation/number of live pups born)x100

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
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality was observed.
One male in 100 mg/kg bw/day group lost hair of loin between day 8 and 47. One female of 100 mg/kg bw/day lost hair between 8 and 17 days of administration and between 0 and 19 days of festation. Another female (not pregnant) of 100 mg/kg bw/day group lost also hair from 21 to 37 days of administration.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Decrease in body weight was observed in both sexes of 100 mg/kg bw/day group until 8 days of administration. Thereafter, animals started to gain but the suppression of body weight gain was noted for a whole administration period.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Decreases in estrus count and prolonged estrous cycle were found in 100 mg/kg bw /daygroup.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
One couple was not mated and two dams were not pregnant in 100 mg/kg bw/day. However, no significant differences were found as for copulation index and fertility index. Decreased duration of mating observed in 4 mg/kg bw/daywas not considered as compound-related effect, since no changes were found in the higher dosing groups.
Increased gestation days and decrease (or trend of decrease) in number of corpora lutea, number of implantations, gestation index, number of litter, number of live new borns and birth index were observed in females of the 100 mg/kg bw/daygroup. No effects related to the test article were observed for the implant index.
All embryo absorption was observed in three dams of the 100 mg/kg bw/daygroup and number of corpora lutea was not examined. Test item could induce the early embryonic lethal.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Relative and the absolute weight of the adrenals increased in males of 20 mg/kg bw/day and more groups. Relative and the absolute weight of thymus decreased in males of 100 mg/kg bw/day group. In 100 mg/kg bw/day group, the absolute weight of heart, thyroids, kidneys, epididymides decreased, and the relative weight of brain, lungs, liver, spleen and testes increased.
In females of 100 mg/kg bw/day group, the absolute weight of heart, thymus and ovaries decreased, and the relative weight of brain, lung, liver, spleen and kidneys increased. In addition, the absolute weight of heart decreased in females of 20 mg/kg bw/day group.
Changes in brain, lungs, heart and thyroids were observed only in absolute weight and there was not correspondent histopathological findings. Therefore, this change didn't have a toxicological meaning.

GROSS PATHOLOGY (PARENTAL ANIMALS)
One male in control group and three males of 100 mg/kg group showed black red spot in glandular stomach. One male of 100 mg/kg bw showed rough surface of kidney. Four males of 100 mg/kg bw showed enlargement of testis. In addition, one male of both control and 100 mg/kg bw group showed diaphragm hernia of liver, one male of 100 mg/kg bw group showed light gray macule of lung, one male of control group showed a white spot in testis, one male of 100 mg/kg bw group showed light gray nodule of testis, one male of control and 20 mg/kg bw group showed light gray nodule of epididymis.
One female of 100 mg/kg group showed atrophy of thymus at the terminal sacrifice. One female which was imminently sacrificed due to death of all newborns showed black red spot in mucosa of glandular stomach and light gray coloration of kidney. Three animals of 100 mg/kg bw group which were non-delivery showed no abnormalities. No abnormalities were also found in two non-pregnant females of 100 mg/kg bw group.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Two males and females of 100 mg/kg bw/day group showed centrilobular hypertrophy. One female of 100 mg/kg bw/day showed vacular degeneraion of hepatocyte. Nine males and six females showed basophilic material in bile duct. Two animals of both sexes in 100 mg/kg bw/day showed deposits of hemosiderin in the Kupffer cells in the liver. In glandular stomach, one male of control group and three males of 100 mg/kg bw/day showed erosion. This effect suggested that test item could cause toxicological effect on glandular stomach. One female of 100 mg/kg bw/day showed ulcer in glandular stomach. In the kidney, necrosis of the tubules in one female of 100 mg/kg bw/day group, basophilic tubules dilatation of tubules in two males of control and 20 mg/kg bw/day and all animals in both sexes of 100 mg/kg bw/day, infiltration of lymphocytes in nine males and eight females of 100 mg/kg bw/day, infiltration of neutrophil in one female of 100 mg/kg bw/day, dilatation tubule in one male and female of 100 mg/kg bw/day were noted. In the spleen, extramedullary hematopoiesis in three males and nine females and deposits of hemosiderin in the red pulp in all animals of both sexes of 100 mg/kg bw were noted. In the femur, decreased hematopoiesis was noted in eleven males of the 100 mg/kg bw group. In the thymus, atrophy was noted in one male and for females of 100 mg/kg bw/day group. In adrenal, necrosis of cortex was noted in one female of 100 mg/kg bw/day. The change in adrenal was not considered as a compound-related effect but it was caused by the stress of pregnant and delivery. In addition, no changes like this were found in other females and males. In the testis, dilatation of seminiferous tubule in four males, vacuolization of the Sertoli cells in two males, degeneration of germ cells in one male, multinucleated giant cells in one male were note in 100 mg/kg bw/day group. In the epididymis, infiltration of lymphocytes in two males, germ cell debris in the lumen in one male, edema in one male were noted in 100 mg/kg bw/day group. These effects on testis and epididymis were may be caused by the circulation disorder since these changes are similar to the effects caused by administration of cadmium chloride. However, fertility was not affected by the test item in males.
In addition, necrosis of hepatocyte, accumulation of foam cell, mineralization of artery, hemorrhage of lungs and thymus, myocardial degeneration, atrophy of seminiferous tubule, granuloma of spermatic of testis and epididymis were regarded as not relevant toxicological effect but changes caused naturally.

OTHER FINDINGS (PARENTAL ANIMALS)
Bone marrow examination showed decreases in nucleated cell counts and the M/E ratio, and decrease in cell counts of neutrophils, eosinophils, lymphocytes, basophilic erythroblasts, and polychromatophilic and orthochromatophilic erythroblasts in males of the 100 mg/kg bw/day group. Test item could inhibit hematopoiesis.

Effect levels (P0)

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Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Decrease in body weight and food consumption in males and females. Effect on hematology and clinical chemistry in males.
Dose descriptor:
NOEL
Remarks:
reproduction
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse or treatment-related effects on reproductive performance were observed up to and including the highest tested dose level
Dose descriptor:
NOEL
Remarks:
reproduction
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: decreases in estrus count, numbers of corpora lutea and implantation and birth index, and an extension of the estrous cycle and increased gestation days were observed in females at 100 mg/kg bw/day

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
no external anomalies
Histopathological findings:
not specified

Details on results (F1)

VIABILITY (OFFSPRING)
One dam didn't collect pups after delivery in 100 mg/kg. Therefore, still birth index was increased. However this change was not considered as a compound-related effect because other animals didn't show such changes.
No differences were found between treatment group and control group for viability index.

CLINICAL SIGNS (OFFSPRING)
No effects related to the test article were observed for sex ratio of live newborns.

BODY WEIGHT (OFFSPRING)
No differences were found between treatment group and control group as body weight of live newborns at day 0 and 4.

GROSS PATHOLOGY (OFFSPRING)
No external anomalies were also found in all groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse or treatment-related effects were observed up to and including the highest tested dose level

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion