Registration Dossier

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:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

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:
not specified
GLP compliance:
yes
Limit test:
no
Justification for study design:
Investigation of the repeated-dose, reproductive and developmental toxicity of the test substance as well as the reversibility of the potential toxic changes.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Appearance: white powder with a characteristic faint odor

Test animals

Species:
rat
Strain:
other: Crj:CD(SD)IGS
Sex:
male/female
Details on test animals and environmental conditions:
- Seventy each of 8-week-old male and female Sprague-Dawley SPF rats (Crj;CD (SD) IGS, Charles River Laboratories Japan, Inc., Atsugi Breeding Center) were purchased (number of animals received: 73 each of males and females)
- The animals were quarantined and acclimatized for 14 days. During this period, the general condition, body weight, and estrous cycle (for 9 days after the quarantine period) were examined, and 58 each of males and females without any abnormality of the general condition (males) (or without any abnormality of the general condition or estrous cycle and with good weight increase for females) were selected and administered the test substance at the age of 10 weeks.
- The body weight at the start of the test substance administration ranged from 338 to 395 g for the males and 219 to 256 g for the females.
- Animals were housed individually in a bracket type metal wire cage in an animal breeding room maintained at a temperature of 21°C to 26°C, relative humidity of 37% to 77%, ventilation frequency of 10 to 15 air changes per hour, and illuminated for 12 hours per day (07:00 to 19:00).
- The animals were allowed free access to Solid chow NMF (nonsterilized: Oriental Yeast Co., Ltd., batch numbers 040713, 040806, 040913) from a stainless steel feeder and to tap water (city water of Gotenba, water bottle was used).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
The test substance was administered by oral gavage, a method commonly employed for oral administration to rodents. Each animal received 5 mL/kg body weight of the test suspension by forced oral administration via a gastric tube (08:20-14:24 h). Animals of the control group received the vehicle (olive oil) in a similar manner. The volume of the test suspension was calculated based on the latest body weight of each animal.
Details on mating procedure:
- After the end of the pre-mating administration period, each pair of male and female animals in the same dose group of the main group was housed in the same cage overnight, and the females were judged to have copulated if the vaginal smear contained sperm or the presence of the vaginal plug was confirmed. Days to copulation was calculated from the day of mating, taken as day 0. From gestation day 17 to day 5 of lactation, the animals were housed individually in a plastic Econ cage with bedding.
- Observation of mother animals. All female animals confirmed to have copulated were allowed to undergo spontaneous delivery, and observed for the presence/absence of abnormalities in the delivery. Delivery completion was checked twice daily (morning, afternoon) from gestation day 21 to the morning of gestation day 25, from which the gestation period was calculated in units of 0.5 days. If delivery was complete by 5:00 h in the afternoon, that day was regarded as day 0 of lactation.
- Mother animals that completed the delivery were observed for the presence/absence of pup licking and ingestion of the placenta and amnion. They were allowed to suckle pups up to day 4 of lactation (the date of delivery completion was regarded as day 0 of lactation) and observed for lactating behavior, using pup gathering, nest building, and breastfeeding as indices.
Analytical verification of doses or concentrations:
yes
Remarks:
HPLC
Details on analytical verification of doses or concentrations:
- An appropriate amount of the test substance for each concentration was weighed and suspended in olive oil in an agate mortar to prescribed concentrations (50, 100, and 200 mg/mL). The test suspensions were prepared at a frequency of at least once every 7 days and stored in a cold dark place (refrigerator, observed temperature: 3°C-5°C) in light-protected containers (brown glass bottles) until use.
- Stability of the test suspensions was confirmed at Bozo Research Center and showed that 50 and 200 mg/mL suspensions of the test substance (vehicle: olive oil) remained stable for 24 hours at room temperature after storage in light-protected containers in a cold dark place (refrigerator) for 7 days.
- Confirmation of the concentrations and uniformity of the test suspensions of each concentration used for administration at week 1 and on the last week of administration were analyzed by HPLC at the Bozo Research Center. The results showed that for all the suspensions tested, the percentage of the test substance relative to the nominal value was in the range of 96.5% to 105.0%, with a C.V. in the range of 1.0% to 5.3%, which were within the acceptable range (concentration, nominal value ± 10%; C.V., ≤ 10%).
- Analytical method:
The test sample (dosing suspension), 1 mL, was diluted with 60 vol% of THF solution to 10 mL and centrifuged (2000 rpm, 1000 × g, 20°C, 5 minutes); then, 1 mL of the lower layer was diluted to 5 mL with the mobile phase of HPLC. The diluted solution was filtered through a Milex HV filter and the filtrate was subjected to measurement by the HPLC system. Single test samples were taken from the upper, middle and lower layers of the dosing suspension.
Duration of treatment / exposure:
The duration of administration was 14 days before mating, 14 days during the mating period, and 14 days after the mating period, that is, 42 days in total, for the males of the main group and the males and females of the recovery group, and 14 days before mating and up to day 4 of lactation throughout the mating and gestation periods, that is, 42 to 45 days in total, for the females of the main group. The recovery period for the males and females of the recovery group was 14 days after the end of administration, during which period the test substance was not given to the animals.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 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)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
A total of 4 dose groups were set up: 250, 500, and 1000 mg/kg bw groups and the control group. Each main group consisted of 12 male and female animals each, and each recovery group consisted of 5 each of males and female animals in the control and high- dose groups.
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for selection of doses. In a previous study “14-day repeated-dose oral toxicity study of the test substance in rats (a preliminary study)” (doses: 125, 250, 500, and 1000 mg/kg bw, Bozo Research Center study No.: C-R016), administration of the test substance did not produce any effect even at 1000 mg/kg bw, the level defined as the limiting dose by the OECD Guideline for Testing of Chemicals 422. Therefore, 1000 mg/kg was set as the highest dose, and doses of 500 and 250 mg/kg bw were derived by dividing by a common factor of 2. A total of 3 doses were thus set up.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
- Observation of the general condition. All animals were observed for the presence of any abnormality of the general condition, such as in the external appearance, nutritional condition, posture, behavior, and excrements, 3 times everyday (before, immediately after, and 2 hours after the administration) during the administration period and once every morning during the recovery period.
Detailed observation of the general condition, function tests, measurement of the grip strength and spontaneous motor activity. Detailed observation of the general condition was performed once before the start of administration for all animals, once every week during the administration period for the males of the main group, once every week during the pre-mating administration and mating periods, and on predetermined days (gestation days 1, 7, 14 and 20, day 4 of lactation) during the gestation period and the lactation period for the females of the main group, and once every week during the administration and recovery periods for the animals of the recovery group. Function tests, measurement of the grip strength, and measurement of the spontaneous motor activity were performed on the last week of administration (day 39 of administration) for the males of the main group, after F1 necropsy on day 4 of lactation (day 42-45 of administration) for the females of the main group, and on the last week of administration (day 39 of administration) and last week of the recovery period (day 11 of recovery) for the males and females of the recovery group. These tests were performed on 5 animals each per group. - Measurement of body weight. Body weight was measured on days 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39 and 42 of administration, and on the day of necropsy for the males of the main group, on days 1, 4, 8, 11 and 14 of the recovery period and on the day of necropsy, in addition to the days of measurement for the males of the main group, for the males and females of the recovery group, and on days 1, 4, 8, 11 and 15 of administration (and days 18, 22 and 25 of the administration period as well as in non-copulated animals), days 0, 4, 7, 11, 14, 17 and 20 of gestation, and days 0 and 4 of lactation for the females of the main group. Body weight was measured from 08:06 to 10:45 h, except for the measurement on day 0 of lactation for females whose end of delivery was confirmed during the observation in the afternoon. On the day of necropsy, the body weight was measured after the animals had been denied access to food for approximately 16 hours from the previous day, in order to calculate the relative organ weight.
- Measurement of food consumption. The amount of food remaining relative to that supplied on the previous day was measured on days 1, 4, 8, 11, 15, 32, 36, 39 and 42 of administration for the males of the main group, on days 1, 4, 8, 11 and 14, in addition to the days of measurements for the males of the main group, for the males and females of the recovery group, on days 1, 4, 8, 11 and 15 of administration, days 1, 4, 7, 11, 14, 17 and 20 of gestation, and days 2 and 4 of lactation for the females of the main group. Food consumption per animal was calculated from the data thus obtained. The amount of food supplied and the amount of food remaining were measured from 08:26 to 11:25 h.
- Urinalysis (including measurement of water intake). On the last week of administration (day 36 to 37 of administration) and on the last week of the recovery period (day 8 to 9 of recovery), each of the male animals was individually housed in a cage equipped with a urine collector. Four-hour urine specimens were collected under fasting conditions of the animals with free access to water, followed by 20-hour urine specimen collection under free access to food and water. The parameters tested are as shown below. The first 4-week urine specimens were subjected to tests from pH up to sediments, as well as measurement of the urine volume, and the subsequent 20-hour urine specimens were subjected to measurement of the osmotic pressure and urine volume. Urine volume was calculated as the sum of the volumes of 4-hour and 20-hour urine specimens. The amount of water intake from the previous day was measured using a water bottle while the animals were housed in the cage equipped with the urine collector.
Oestrous cyclicity (parental animals):
Vaginal smear was collected everyday from all females of the main group, from day 1 of administration until copulation was confirmed, and subjected to microscopic examination. During the pre-mating period, the vaginal smear profile was classified into proestrus, estrus, metestrus, and anestrus, from which frequency of the estrus and the days from one estrus to the next estrus (estrous cycle) were calculated. During the mating period, vaginal smears were examined to confirm the presence/absence of sperms.
Sperm parameters (parental animals):
-
Litter observations:
- The number of live pups and the number of stillborn pups were counted on the day of birth. Live pups were observed once everyday up to day 4 of lactation. Live pups were also observed for the presence/absence of any external abnormalities, checked for sex, their body weight was measured, and they were allowed to suckle the mother. Pups with external abnormalities were fixed in 10 vol% formalin solution and stored.
- External anomaly rate (%) = (number of pups with external anomalies/total number of pups) × 100
- Sex ratio = (number of male pups)/(number of male plus female pups)
Postmortem examinations (parental animals):
After delivery, the mother animals were exsanguinated to death by dissecting the abdominal aorta on day 5 of lactation, after the animals had been denied access to food overnight (approx. 16-20 h) from day 4 of lactation: 5 in each group after blood collection for hematological tests and blood chemistry tests, and the remaining animals under ether anesthesia.
- Necropsy and organ weight measurement. All of the 5 male and female animals in each group from which blood samples were collected for the hematology and blood chemistry tests on the day after the last day of administration and on the last day of the recovery period were exsanguinated to death after the blood collection, and all the other animals were exsanguinated to death by dissecting the abdominal aorta under ether anesthesia. They were then subjected to detailed gross pathological examination of the body organs and tissues, including the external surface, head, chest and abdomen, and the results were recorded. In the females (mother animals), the number of corpora lutea and number of implantation sites were counted on day 5 of lactation. Then, in 5 each of the male and female animals from which blood samples were collected for the hematology and blood chemistry tests, the weight (absolute) of the following organs (testes and epididymes of all the animals) was measured and the relative weight of each organ per 100 g body weight was calculated from the absolute organ weight and the body weight at necropsy. For bilateral organs marked with an asterisk, the weight of each side was measured separately and the sum of the weights was calculated. Brain, thyroid gland* (including parathyroid gland), thymus gland, heart, liver, spleen, kidney*, adrenal*, testis*, and epididymis*.
- Histopathological examination. The following organs and tissues of all the animals were fixed and stored in 10 vol% formalin solution in phosphate buffer (the testes and epididymes were fixed in Bouin's fluid, followed by storage in 10 vol% formalin solution in phosphate buffer). Then, organs and tissues (see below) were embedded in paraffin, and sections were stained with hematoxylin and eosin (H-E). Specimens obtained from 5 each of the male and female animals of the control and high-dose groups from which blood specimens were collected for the hematology and blood chemistry tests were subjected to microscopic examination (for bilateral organs, both sides were isolated and one side was subjected to the microscopic examination). The results revealed the effect of the test substance on the stomach. Therefore, specimens from 5 each of the male and female animals of the low- and medium-dose groups were also subjected to microscopic examination. Representative cases of normal and abnormal findings were photographed.
(Cerebrum, cerebellum, pituitary gland, spinal cord (thoracic), sciatic nerve, thyroid gland, parathyroid gland, adrenal, thymus gland, spleen, submandibular lymph nodes, mesenteric lymph nodes, heart, lung (including bronchus), stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, kidney, bladder, testis, epididymis, ovary, uterus, seminal vesicle, sternum (including bone marrow), femur (including bone marrow), and the animal identification site (auricle))
Postmortem examinations (offspring):
After the body weight was measured on day 4 of lactation, all the pups were exsanguinated to death under ether anesthesia and necropsied to examine for the presence/absence of abnormalities in the organs and tissues, including the head, chest, and abdomen. Body weight was measured for individual pups and the mean body weight was calculated separately for each of the male and female littermates
Statistics:
- Bartlett test
- Dunnett’s test
- χ2 test with Yates’ continuity correction
- Fisher’s exact test
Reproductive indices:
Copulation rate (%) = (number of pairs that copulated/number of pairs) × 100
Conception rate (%) = (number of pregnant females/number of females that copulated) × 100
Fertility rate (%) = (number of pregnant females/number of males that copulated) × 100
Gestation period (days) = Day 0 of lactation - day 0 of gestation
Delivery rate (%) = (number of females giving birth to live pups/number of pregnant females) × 100
Implantation rate (%) = (number of implantation sites/number of corpora lutea) × 100
Offspring viability indices:
Stillbirth rate (%) = (number of stillborn pups/total number of pups) × 100
Live birth rate (%) = (number of live-born pups/total number of pups) × 100
Viability rate of live-born pups (%) = (number of pups alive on day 4 of lactation/number of pups alive on day 0 of lactation) × 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- In the main group, one female in the 500 mg/kg bw group showed opacity of an eyeball (unilateral) from gestation day 5, which was not related to the dose and was therefore considered to be an incidental change.
- In the recovery group, one male in the 1000 mg/kg bw group showed decreased spontaneous motor activity from day 37 of administration up to day 7 of the recovery period, and wheezing from day 37 of administration until the end of the recovery period.
- No abnormality was observed in the other animals, either in the main or in the recovery groups.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no significant difference in the body weight between males and females of the main group. A significantly greater increase in body weight was observed in the females of the 250 and 1000 mg/kg bw groups during the lactation period, but the increase was not dose-related.
- In the recovery group, males in the 1000 mg/kg bw group showed decreased body weight gain during the administration period and decreased body weight (-21g) during the recovery period. This was caused by the abnormality in 1 out of the 5 animals. This animal showed continued body weight decrease (and also decreased spontaneous activity and wheezing in the observation of the general condition; the body weight was 466g before manifestation of the symptom and 261g on day 14 of the recovery period). The body weights of the other 4 males and 5 females in the same group were similar to those of the animals in the control group, showing no statistically significant differences.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Administration of the test substance did not have any effect on the food consumption in the males or females of either the main group or the recovery group. A significant increase was observed on days 2 and 4 of lactation in the females of the 250 mg/kg dose group in the main group, but this was not dose-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
-Tests at the end of the administration period. A significant increase in the serum level of fibrinogen was observed in the females of the 250 mg/kg bw group and a significant decrease in the percentage of lymphocytes and significant increase in the percentage of segmented neutrophils were observed in the females of the 500 mg/kg bw group. However, none of these changes were observed in the 1000 mg/kg bw group, suggesting that they were within the range of physiological variations. No significant differences were observed in the male animals between the control group and any of the treatment groups.
- Tests at the end of recovery period. A significant increase in the mean corpuscular volume of the red blood cells, significant decrease of the platelet count, significant increase in the percentage of lymphocytes, and significant decrease in the percentage of segmented neutrophils were observed in females of the 1000 mg/kg bw group. However, these changes were not observed at the end of the administration period, which suggested that they were within the range of physiological variations. No significant differences were observed in the male animals between the control group and any of the treatment groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
- Test at the end of the administration period. A significant increase in the serum level of ALT was observed in the males of the 1000 mg/kg bw group. A significant decrease of the serum level of inorganic phosphorus was observed in the males of the 250 mg/kg bw group. However, since the decrease was not dose-related, it was considered to be within the range of physiological variations.
- Tests at the end of recovery period. A significant increase in the serum level of total protein was observed in the females of the 1000 mg/kg bw group. However, since no such change was observed at the end of the administration period, the increase was considered to be within the range of physiological variations.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis (including measurement of water intake) showed no abnormalities in the qualitative parameter values in any of animals in either the main group or in the recovery group. No significant difference was observed in any of the quantitative parameter values between the control group and any of the treatment groups.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Observation of animals in the home cage. No abnormalities were observed in any of the animals in either the main group or the recovery group.
- Observation of the animals while being handled. No abnormality was observed in any of animals in either the main group or the recovery group.
- Observation of animals in the open field. Males of the 1000 mg/kg bw group in the main group showed a significant increase of the defecation frequency during weeks 1 and 2 of administration, which was a very mild transient change and considered to be within normal range. No abnormalities were observed in the other parameters in any of the animals in either the main group or the recovery group. No significant differences were observed in the standing frequency between the control group and any of the treatment groups.
- Function tests. No abnormalities were observed in any of the animals in either the main group or the recovery group. No significant differences were observed in the air righting reflex or landing foot splay between the control group and any of the treatment groups.
- Measurement of the grip strength. No significant differences were observed between the control group and any of the treatment groups in either the main group or the recovery group.
- Measurement of spontaneous motor activity (measured for 10-minute periods and a total of 60 minutes). No significant difference was observed between the control group and any of the treatment groups in either the main group or the recovery group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Administration of the test substance had effects on the stomach of the animals in the 250 mg/kg bw and higher dose groups.
- Findings at the end of the administration period. Stomach: Mild to moderate erosions or ulcers of the anterior stomach were observed in 0, 4 and 4 males and 1, 1 and 1 females of the 250, 500, and 1000 mg/kg bw groups, respectively. Very mild to moderate thickening of the anterior stomach mucosa was observed in 1, 4 and 5 males and 1, 4 and 3 females of the 250, 500, and 1000 mg/kg bw groups, respectively. Very mild to mild edema of the submucosal tissue in the anterior stomach was observed in 1, 5 and 5 males and 0, 4 and 3 females of the 250, 500, and 1000 mg/kg bw groups, respectively. Most of these changes in the anterior stomach were localized findings. All of the other findings observed, as follows, were considered to be incidental changes as judged from the frequency of their occurrence and the histopathological findings (Epididymis: Very mild infiltration by stromal cells was observed in 1 male of the control group. Heart: Very mild localized myocarditis was observed in 4 males of the control group and 1 male of the 1000 mg/kg bw group. Kidney: Very mild basophilic tubules were observed in 3 males of the control group and 1 male and 1 female in the 1000 mg/kg bw group. Liver: Very mild, minute granulomas were observed in 3 males of the control group and 1 male of the 1000 mg/kg bw group. Lung (including bronchi): Very mild mineral deposits in the arterial walls were observed in 1 male of the control group and 1 female of the 1000 mg/kg bw group. Very mild accumulation of foam cells was observed in 2 males and 1 female of the control group, and 1 male and 3 females of the 1000 mg/kg bw group. Spleen: Very mild to mild extramedullary hematopoiesis was observed in 5 females each in the control group and 1000 mg/kg bw group. Stomach: Inclusion cysts were observed in 1 male of the 500 mg/kg bw group. Very mild to mild erosions in the glandular stomach were observed in 0, 0, 0 and 1 male and 3, 1, 1, and 0 females in the control group, 250, 500, and 1000 mg/kg bw group, respectively).
- Findings at the end of the recovery period. Stomach: Moderate thickening of the anterior stomach mucosa was observed in 1 male of the 1000 mg/kg bw group.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no animals that showed abnormal estrous cycles. No significant differences were observed in the mean length of the estrous cycle between the control group and any of the treatment groups.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- Mating results. One pair in the 500 mg/kg bw group did not copulate, whereas copulation occurred in all of the other pairs by day 4 after the start of mating, resulting in pregnancy in all of the females that copulated. Thus, there were no significant differences in the days to copulation, copulation rate, fertility rate, or conception rate between the control group and any of the treatment groups.
- Delivery results and delivery/lactating state. All pregnant females delivered normally from day 21.5 to 23.0 of gestation. After administration of the test substance, there were no significant difference in the delivery rate, gestation period, number of corpora lutea, number of implantation sites, implantation rate, stillbirth rate, number of live-born pups, and live birth rate. Significant increases in the number of corpora lutea and in the live-born pups were observed in the 250 mg/kg bw group, but they were not dose-related. In regard to nursing, no abnormalities were observed in nest building, pup gathering, or lactating behavior in any of the mother animals.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed on reproductive parameters
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant systemic adverse effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
During the lactation period, death occurred in only 4 pups in the control group and 2 pups in the 1000 mg/kg bw group. Thus, there were no significant differences in the viability rate on day 4 of lactation between the control group and any of the treatment groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences were observed in the body weight of the males or females at birth or on day 4 of lactation between the control group and any of the treatment groups.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex ratio: no effects
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Thymic cervical residue was observed in the 1, 1, 0 and 2 males and 3, 1, 0 and 3 females of the control group, 250, 500, and 1000 mg/kg bw groups, respectively, however, the occurrences were not dose-related.
- No significant differences in external anomalies rate were observed. A kinking of the tail in 1 animal of the 500 mg/kg bw group was recorded, but was considered to be a spontaneous occurrence as judged from the frequency of occurrence and the type of the anomaly.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Repeated-dose toxicity:

Administration of the test substance had no effect on any of the following: detailed observation of the general condition, results of function tests, grip strength, spontaneous motor activity, body weight, food consumption, or the results of urinalysis (including water intake), or hematological tests. In regard to the general condition, decreased spontaneous motility, wheezing, and decreased body weight were observed in 1 male of the 1000 mg/kg bw group in the recovery group. Gross examination showed rough mucosa in the anterior stomach and expansion of the digestive tract from the stomach to the colon due to gas accumulation, and histological examination showed thickening of the gastric mucosa. Such findings were observed in only 1 animal. Body weight in this animal showed a similar increase as that observed in the control group until the above symptoms manifested themselves on day 37 of administration, but continuously decreased after the manifestation of these symptoms, from which these symptoms were judged to be due to inadvertent administration of the test substance into the upper respiratory tract. In regard to the blood chemistry tests, a significant increase of the serum ALT was observed in the males of the 1000 mg/kg bw group at the end of the administration period. Since the findings were very mild and within the range of physiological variations and not accompanied by histopathological abnormalities, it was judged that these findings were not caused by the test substance. In the pathological examination performed at the end of the administration period, gross observation showed indentation of the anterior stomach in the animals of the 250 mg/kg bw and higher dose groups, and rough mucosa and/or white foci in the anterior stomach in the animals of the 500 mg/kg bw and higher dose groups, and histological observation showed erosions/ulcers of the anterior stomach, thickening of the anterior stomach mucosa, and edema in the submucosal tissue, suggesting the irritability of the test substance.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the systemic and reproductive rat NOAELs (P0 generation) of the test substance were determined to be <250 and 1000 mg/kg bw/day, respectively and the rat NOAEL (F1 generation) was determined to be 1000 mg/kg bw/day.
Executive summary:

A study was conducted to determine the toxicity to reproduction of the test substance according to OECD Guideline 422, in compliance with GLP. The test substance was administered at 0 (control group), 250, 500 or 1000 mg/kg bw to male Sprague-Dawley SPF rats for 14 days before mating, through the mating period, and up to 1 day before necropsy (42 days in total) and to female Sprague-Dawley SPF rats for 14 days before mating, through the mating period and the gestation period, up to day 4 of lactation (42 to 45 days in total) to investigate the repeated-dose, reproductive and developmental toxicities. In the 0 and 1000 mg/kg bw groups, a 14-day recovery period was allowed after the 42-day administration period to investigate the reversibility of the toxic changes. No test substance-related effects were observed regarding clinical signs, detailed clinical findings, function tests, grip strength, amount of spontaneous movement, body weights, food consumption, urinalysis (including water intake), hematology or blood chemistry parameters. Gross pathological examination at the end of the administration period revealed recessed areas in the forestomachs at 250 mg/kg bw and above and rough mucosa or white foci at 500 mg/kg bw and above, and erosion/ulceration, mucosal thickening and submucosal edema at 250 mg/kg bw and above on histopathological examination. Administration of the test substance did not have any effect on the estrous cycle, days to copulation, copulation rate, fertility rate, or conception rate. Similarly, administration of the test substance did not have any effect on the delivery rate, gestation period, number of corpora lutea, number of implantation sites, implantation rate, stillbirth rate, number of live-born pups, live-birth rate in the mother animals, or on the sex ratio of the littermates. No abnormalities were observed in the lactating behavior during the lactation period either. These results suggest that administration of the test substance even at 1000 mg/kg bw had no effect on the reproductive function, such as that shown by the copulation rate, of the males or females, or in the fertility rate, conception rate, or on the gestation maintenance, delivery, or lactating behavior in the mother animals. Pups showed no changes caused by the administration of the test substance regarding the observation at birth, necropsy findings on day 4 of lactation, body weight, or viability rate, which suggested that administration of the test substance even at 1000 mg/kg bw had no effect on the development. Under the study conditions, the rat NOEL of the test substance was determined to be <250 mg/kg bw/day for parental systemic toxicity in both males and females, and 1000 mg/kg bw/day for reproductive and developmental toxicity in both male/female parent animals and pups.