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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 4, 1997 – August 2, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study according to OECD guideline 422

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD combined repeated dose and reproductive/developmental toxicity screening test
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of divinylbenzene and ethylstyrene
EC Number:
910-757-7
Cas Number:
N/A
Molecular formula:
Divinylbenzene: C10H10 Ethylstyrene: C10H12
IUPAC Name:
Reaction mass of divinylbenzene and ethylstyrene
Details on test material:
Source: Shin Nippon Steel Chem.
- Name of test material (as cited in study report): divinyl benzene
- Molecular weight: 130.19
- Melting point: -29°C
- Boiling point: 190–200°C
- Physical state: At ordinary temperatures, it is a colorless or pale yellow transparent liquid.
- Analytical purity: purity 96.2%; impurities: 3.2 wt% ethyl vinyl benzene, 1010 ppm P-tert-butyl catechol, 0.6 wt% other
- Purity test date: 96.3%
- Lot/batch No.: Lot No. SXS2
- Other: In this test, the substance provided by the Environmental Chemical Safety Countermeasures Section, Planning Department, Environmental Health Bureau, Ministry of Health and Welfare, on October 26, 1996 (manufactured by Nippon Steel Chemical Group Co., Ltd.)

- Control article: Corn oil [Katayama Chemical Industries Co., Lot No. 1726 (obtained on January 8, 1997; limit on period of use: January 7, 2002) and Lot No. 1726 (obtained on April 10, 1997; limit on period of use: April 9, 2002)] was obtained. It was stored in the storeroom of the Test Substance Storage Department of the test center.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals or test system and environmental conditions:
Sprague-Dawley male and female rats [(SPF), Crj:CD(SD)IGS], were used in this test. Seventy-three males and the same number of females were purchased on April 9, 1997 from the Charles River Japan Co. (Hino Rearing Center) at 8 weeks of age. The body weight ranges one day after they were obtained were 249–290 g for the males and 18–217 for the females
TEST ANIMALS
- Source: Charles River Japan Co. (Hino Rearing Center)
- Age at study initiation: 10 weeks for both the males and females
- Weight at study initiation: The body weight ranges were 315–352 g for the males and 211–239 g for the females.
- Fasting period before study: not reported
- Housing: During the quarantine and acclimation periods, the animals were kept in stainless steel suspended-type cages (W: 240 x D: 380 x H: 200 mm), up to 5 to a cage. After the division into groups, they were reared individually in stainless steel cages (connected in groups of 5) (W: 755 x D: 210 x H: 170 mm). However, for the mating stainless steel suspended cages were used. The dams were transferred, on the 18th day of pregnancy, to individual plastic cages (W: 310 x D: 360 x H: 175 mm) with autoclave-treated floor covering (Sanfureeku, Charles River Japan Co.), and allowed to give birth and nurse naturally. The drip trays, water bottles, and plastic cages were changed 2 or more times a week and the stainless steel cages connected in groups of 5 and the food supply devices were changed once every two weeks or more.
- Diet: ad libitum - solid food (CRF-1, Oriental Yeast Industries Co.)
- Water: ad libitum - Tap water was supplied to the animals using water bottles. The results of testing the water quality were obtained from the Ibaraki Prefecture Public Health Test Center approximately every 3 months.
- Acclimation period: 7-day acclimation period. During these periods, 3 body weight measurements and daily observations of their general conditions were performed, and the estrus periods of the females were observed for 7 days during the acclimation period. If no abnormalities were seen in the general conditions and the changes in the body weights, and no abnormalities were observed in the estrus observation, the animals were divided into groups and used in the test.

During the quarantine and acclimation periods, the animals were identified by using a method of writing with oil-based ink and a hair dyeing method with a colored dye on the day they were obtained, and after they were divided into groups, [the groups] were identified with a hair dyeing method with a colored dye and an ear-punching method. Furthermore, during the quarantine and acclimation periods, each cage was identified with a label showing the test number, date of obtaining of the animals, sex, and quarantine/acclimation period number; after the division into groups, each cage was identified with a label showing the test number, dose, sex, and animal numbers.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): range of 20–24°C (actually measured values: 20–24°C)
- Humidity (%): setting of 40–70% (actually measured values: 43–59%)
- Air changes (per hr): 12 hours light and 12 hours darkness (light: 6:00 a.m.–6:00 p.m.)
- Photoperiod (hrs dark / hrs light): 12 air exchanges/hour (fresh air with bacteria filtered out)

IN-LIFE DATES: From: April 4, 1997 To: August 2, 1998

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Oral administration was selected as the administration route for the divinyl ben-zene because it is believed that it can be continuously ingested orally by human beings. Vehicle ws corn oil.
- Concentration in vehicle:
Group Test group Dose (Concentration)
_________________________________________________________________________
Group 1 Control 0 mg/kg (0 mg/mL)
Group 2 Divinyl benzene 30 mg/kg
Group 3 Divinyl benzene 100 mg/kg
Group 4 Divinyl benzene 300 mg/kg
Group 5 Divinyl benzene 1000 mg/kg
___________________________________________________________________________

- Amount of vehicle (if gavage): The quantity of the liquid administered to the males was calculated as 5 mL/kg based on the body weights measured on the administration day or the day closest to the administration day. For the females, it was calculated as 5 mL/kg, based on the body weights measured on the administration day or the day closest to the administration day during the pre-copulation and copulation periods, the body weights measured on days 0, 7, 14, and 21 of pregnancy during the pregnancy periods, and day 0 of nursing during the nursing period. The administrations were performed once a day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the test substance in the administered samples of the various concentrations which were used on the date of the start of the administrations and the date of the completion of the administration period to the males were measured by using high-performance liquid chromatography at the test center. The results were that the test substance concentrations were in the range of 104.1–1.5.9% of the indi-cated concentrations. These were within the range of suitable concentrations that had been established (±10% of the indicated concentrations); thus, there was no problem with the concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: mated 1-to-1 within the same groups
- Length of cohabitation: The limit of the copulation period was set at 14 days; the animals were allowed to live together and copulate until copulation was verified.
- Proof of pregnancy: The verification of copulation was performed at almost the same time each night; the females in the vaginas of which which semen or vaginal plugs were confirmed were considered to have established copulation, and that day was recorded as day 0 of pregnancy.
Duration of treatment / exposure:
The administration period was determined to be a total of 49 days (14 days pre-copulation and 35 days after it) for the males, and a total of 41–53 days (14 days pre-copulation, a maximum period of 14 days during the copulation period, and the remainder for the pregnancy period and up to the day before autopsies on day 4 of nursing) for the females. The day on which the administrations were started was taken as day 1 of the administrations.
Frequency of treatment:
once daily
Duration of test:
Male, 50 days
Females, day 4 of lactation (41–53 days)
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 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:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were determined according to the results of preliminary tests in which oral administrations to male rats were performed for 2 weeks (doses: 0, 125, 250, 500, and 1000 mg/kg, 5 rats per group). Drooling was observed immediately after the administration in the 125 mg/kg and higher groups, and low values of the body weight and the quantity of food consumed were seen in the 1000 mg/kg group, but no deaths were observed in any group. Therefore, the doses for the test were determined to be 1000 mg/kg, which was considered to be the critical dose, as the highest dose, and the lower doses as 300, 100, and 30 mg/kg, using a ratio of 3, according to the OECD Guideline.

Examinations

Maternal examinations:
The general conditions of the animals and the presence or absence of dead animals were observed twice a day before and after the administrations during the administration period (except for once before the autopsy on the day of autopsy). The body weights were measured twice a week (measurement days: 1, 4, 8, 11, 15, 18, 22, 25, and 29 of administration) for 14 days before the beginning of copulation and during the copulation period, on days 0, 7, 14, and 21 of the pregnancy period, and days 0 and 4 of the nursing period. The measurements of the quantity of food consumed were performed twice a week for 14 days before the copulation period (days on which remaining food was measured: 3, 6, 10, and 13 of administration). Furthermore, the measurements were performed on days 2, 9, 16, and 21 during the pregnancy period and day 4 during the nursing period. The oestrus periods were observed once a day from the first day of the administrations until the day s on which copulation was confirmed. Furthermore, when the estrus periods were observed over 2 days in a row, they were counted as one period.
Ovaries and uterine content:
No data
Fetal examinations:
- External examinations: Yes:
Neonates (F1)
(1) Observation at the time of delivery - The total numbers of delivered pups, the sexes, the numbers of stillborn pups, the numbers of neonates, and the presence or absence of external abnormalities were observed. The stillborn pups were fixed with 20% neutral buffered formalin and stored.
(2) Observation of neonates - The general conditions and presence or absence of deaths of the neonates were observed once a day during the survival period. The dead pups were fixed with 20% neutral buffered formalin and stored.
(3) Body weights - The body weights were measured on day 0 of nursing (date of birth) and day 4 of nursing.
(4) Autopsies - The surviving pups were sacrificed by exsanguination from the abdominal aorta under ether anesthesia and autopsied after the observations on day 4 of nursing were finished. The pups in which abnormalities were observed in the autopsies were fixed in 20 neutral buffered formalin and stored.
Statistics:
Mean values and standard deviations in each group were calculated. The homoscedasticity was tested by Bartlett’s method; in the homoscedastic cases, dispersion analysis was performed by the one-way layout method. If the results were significant, the test was performed by Dunnett’s method. On the other hand, if homoscedasticity was not observed, analysis was performed by the one-way layout method, using ranks (the Kruskal-Wallis test); if the results were significant, Dunnett’s test method using ranks was performed.
Indices:
Numbers of oestrus periods, copulation rates, numbers of days required for copulation, numbers of fertilized females, pregnancy periods, parturition states, nursing states, impregnation rates, numbers of corpora lutea, numbers of implantation traces, implantation rates, birth rates, total number of pups born, delivery rate, stillborn pups, surviving pups on day 4 of nursing and the survival rate on day 4 of nursing, and body weights of neonates
Historical control data:
No data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the fatal case, drooling was observed beginning on day 2 of administration, depilation was observed beginning on day 3 of pregnancy, and lowered skin surface temperature, lowered spontaneous movement, and bleeding from the vaginal mouth were observed beginning on day 15 of pregnancy. In the moribund case, drooling was observed beginning on day 2 of administration and lowered skin surface temperature and lowered spontaneous movement were observed beginning on day 23 of pregnancy.

In the general conditions of the surviving cases, no abnormalities were observed in any of the animal s throughout the observation period in the control and 30 mg/kg groups. In the 100 mg/kg group, drooling was observed in 1–3 cases on days 11–17 of administration, days 0–21 of pregnancy, and days 0–3 of nursing. In the 300 mg/kg group, drooling was observed in 1–12 cases on days 4–18 of administration, days 0–22 of pregnancy, and days 0–3 of nursing. In the 1000 mg/kg group, depilation was observed in one case on days 11–18 of administration, days 0–21 of pregnancy, days 0 and 1 of nursing, depilation was observed in one case on days 15–21 of pregnancy and 0–4 days of nursing, fouling of fur was observed in 1–2 cases on days 4–14 of administration, and drooling was observed in 2–10 cases on days 2–18 of administration, days 0–22 of pregnancy, and days 0–3 of nursing.
Mortality:
mortality observed, treatment-related
Description (incidence):
No deaths or moribund cases were observed in the control, 30, 100, and 300 mg/kg groups. In the 1000 mg/kg group, one animal died on day 17 of pregnancy, and one animal became moribund during delivery on day 23 of pregnancy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights in the 30, 100, and 300 mg/kg groups showed almost the same changes as in the control group before the start of copulation; no significant differences were observed on any measurement days. In the 1000 mg/kg group, significantly lower values of the body weight were seen on days 4–15 of the administration, compared to the control group. In the pregnancy period, the changes in body weight were almost the same in the 30 and 100 mg/kg groups as in the control group; no significant differences were seen on any measurement days. In the 300 mg/kg group, significantly lower values of the body weight, compared with the control group, were seen on days 7 and 14 of pregnancy. In the 1000 mg/kg group, significantly lower values of the body weight, com-pared with the control group, were seen on days 0–21 of pregnancy. Furthermore, in the fatality case in the 1000 mg/kg group, a reduction in body weight was seen before the death. However, no abnormalities were seen in body weight changes during the pregnancy period were seen in the moribund case in the 1000 mg/kg group. During the nursing period, the changes in body weight in the 30, 100, and 300 mg/kg groups were almost the same as in the control group; no significant differences were seen on any measurement days. In the 1000 mg/kg group, significantly lower values of the body weight compared with the control group were seen on days 0 and 4 of nursing.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Before the start of copulation, the quantities of food consumed in the 30, 100, and 300 mg/kg groups were about the same as in the control group; no significant differences were seen on any measurement days. In the 1000 mg/kg group, a significantly low value of the quantity of food consumed compared with the control group was seen on day 3 of administration. During the pregnancy period, the quantities of food consumed in the 30, 100, and 300 mg/kg groups were about the same as in the control group; no significant differences were seen on any measure ment days. In the 1000 mg/kg group, a significantly low value of the quantity of food consumed compared with the control group was seen on day 21 of pregnancy.
During the nursing period, no significant differences in the quantities of food consumed from the control group were seen in the 30 and 100 mg/kg groups. In the 300 mg/kg group, a significantly lower value for the quantity of food consumed, compared with the control group, was seen on day 4 of nursing. In the 1000 mg/kg group, no significant differences were seen compared with the control group, but a tendency for the quantity of food consumed to be low was seen on day 4 of nursing.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the 30 mg/kg group, no significant differences were seen in the absolute or relative weights in any organs, compared with the control group. In the 100 mg/kg group, a significantly low value of the absolute weight of the adrenals compared with the control group was seen, but no constant tendencies were seen in the absolute and relative weights; therefore, this was judged not to be due to the administration. In the 300 mg/kg group, a significantly high value of the relative weight of the kidneys was seen, compared with the control group. Furthermore, in the 300 mg/kg group, significantly low values of the absolute weights of the heart and adrenals were seen, compared with the control group, but since no constant tendencies were seen in the absolute or relative weight s, these changes were judged not to be due to the administration. In the 1000 mg/kg group, significantly low values were seen in the absolute and relative weights of the thymus, a significantly low value of the absolute value of the spleen, and significantly high values of the relative weights of the liver, kidneys, and adrenals, compared with the control group. A tendency toward low values of the relative weight of the spleen was seen, but there was no significant difference. Furthermore, in the 1000 mg/kg group, compared with the control group, significantly low values of the absolute weights of the brain, pituitary, and heart and significantly high values of the relative weights of the brain and ovaries were seen, but these changes showed no constant tendencies in the absolute and relative weights; therefore, they were judged not to be due to the administration.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In the autopsies of the surviving animals, no abnormalities were seen in the control and 300 mg/kg groups. In the 30 mg/kg group, atrophy of the thymus was seen in one case (an impregnated female from which no neonates were obtained). In the 100 mg/kg group, an ulcer of the anterior stomach was seen in one case. In the 1000 mg/kg group, bilateral whitening of the adrenals was seen in one case, and atrophy of the thymus was seen in 7 cases. Atrophy of the thymus was seen in the autopsy of the fatal case of the 1000 mg/kg group. Atrophy of the thymus and dark red spots on the glandular stomach mucosa were seen in the autopsy of the moribund case of the 1000 mg/kg group.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Ulcers in the anterior stomach were seen in one case in the 100 mg/kg group and 2 cases in the 1000 mg/kg group. Their degrees were slight in the 100 mg/kg group and very slight to slight in the 1000 mg/kg group. Eosinophil infiltrations into the ulcer sites were seen in one case in the 100 mg/kg group and 2 cases in the 1000 mg/kg group; their degrees were moderate in the 100 mg/kg group and very slight to slight in the 1000 mg/kg group. Oedema in the ulcer site was seen in one case in the 100 mg/kg group; its degree was moderate. Atrophies of the cortex and medulla of the thymus were seen in 7 cases in the 1000 mg/kg group;
their degrees were slight to moderate. Furthermore, the same kind of change was also seen in the 30 mg/kg group in one case (an impregnated female from which no neonates were obtained) to a moderate degree, but since it was not observed in the 100 or 300 mg/kg groups, it was judged to be a chance change. Atrophy of the marginal zone of the spleen was seen in 2 cases in the 1000 mg/kg group, but its degree was slight. Degeneration and necrosis of the renal tubules in the cortexmedulla boundary of the kidneys was seen in 3 cases in the 1000 mg/kg group; their degrees were slight in all these cases. Furthermore, in the 1000 mg/kg group, significant differences from the control group were observed in the atrophying of the cortex and medulla of the thymus, the atrophy of the marginal zone of the spleen, and the degeneration and necrosis of the renal tubules in the cortex-medulla boundary in the kidneys. Concerning other changes, the following findings were obtained: Focal necrosis of the liver was seen in one case in the control group, but it was very slight. Atrophy of the cortex of the thymus was seen in one case in the 100 mg/kg group, but it was slight. A lymphoma in the spleen was seen in one case in the 30 mg/kg group (an impregnated female from which no
neonates were obtained). Furthermore, these changes were seen only in the control group or in small numbers of cases; therefore, they were judged to be chance changes. In addition, no abnormalities were seen in the control or 1000 mg/kg groups in the hearts, lungs, tracheas, pancreases, duodena, jejuna, ilea, caeca, colons, recta, lymph nodes (submaxillary, mesenteric), adrenals, bladders, ovaries, ovaries, vaginas, pituitaries, thyroids, parathyroids, brains (cerebrum, cerebellum, medulla oblongata), spinal cords, sciatic nerves, bone marrow (sterna, femurs), or mammary glands. Moreover, no changes were seen suggesting the bilateral whitening of the adrenals in one case of the 1000 mg/kg group which were observed in the autopsy. A moderate degree of atrophy of the cortex and medulla of the thymus and a slight degree of atrophy of the marginal zone of the spleen were seen in the fatal case in the 1000 mg/kg group. In the moribund case in the 1000 mg/kg group, a slight degree of central necrosis in the lobules of the liver, a moderate degree of decrease of thymogen granules of the acinar cells of the pancreas, a slight degree of erosion of the glandular stomach mucosa, a moderate degree of atrophy of the cortex and medulla of the thymus, a very slight degree of atrophy of the red spleen medulla, a moderate degree of degeneration and necrosis of the urinary tubules in the cortex-medulla boundary of the kidneys, and a slight degree of reduction of hematopoiesis in the bone marrow were seen.

Maternal developmental toxicity

Other effects:
effects observed, treatment-related
Description (incidence and severity):
Numbers of estrus periods, copulation rates, numbers of days required for copu-lation: No differences were seen between the various administration groups and the control group with regard to numbers of oestrus periods, copulation rates, or number of days required for copulation. In the 1000 mg/kg group, bad development of the mammary glands and bad nest building were seen, and all of the neonates died in the cases of 7/9 dams. Impregnation rates: No differences were seen between the various administration groups and the control group with regard to impregnation rate. Low numbers of corpora lutea and implantation traces were seen in the 1000 mg/kg group. No differences were seen between the various administration groups and the control group with regard to birth rate.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
male reproduction
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
female reproduction
Effect level:
300 mg/kg bw/day (nominal)
Basis for effect level:
dead fetuses
other: Numbers of corpora lutea and implantation scars were decreased in 1000 mg/kg group.

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 30 mg/kg group, significantly high values, compared with the control group, were seen in the separate averages for males and females, the average litter weights, and the total litter weights on day 0 of nursing, but there were no changes which were dependent on the dose. In the 100, 300, and 1000 mg/kg groups, significantly low values, compared with the control group, were seen in the separate averges for males and females, the average litter weights, and the total litter weights on day 0 of nursing. Furthermore, in the 1000 mg/kg group, there were no significant differences from the control group, but there were tendencies towards low values of the separate averages for males and females, the average litter weights, and the total litter weights on day 4 of nursing.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
In the 1000 mg/kg group, a significantly low value, compared with the control group, was seen in the total number of pups born, and a tendency toward a low value of the delivery rate was seen, although there was no significant difference. In the 1000 mg/kg group, significantly low values, compared with the control group, were seen in the number of neonates on day 0 of nursing and the fertility rate, and tendencies to a high value of the number of stillborn pups and a low value of the birth rate of pups were seen, but there were no significant differences. In the 1000 mg/kg group, significantly low values, compared with the control group, were seen in the number of surviving pups on day 4 of nursing and the survival rate on day 4 of nursing.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the observation of the external abnormalities of the neonates, no tail was observed in one case in the 30 mg/kg group and necrosis of the tail was observed in one case in the 100 mg/kg group, but these were judged to be chance occurrences. In the general conditions of the neonates, an external wound on the tail, dropping off of the tail, and lowered surface skin temperature were observed in one case each in the 30 mg/kg group, but these were all judged to be chance occurrences. In the 1000 mg/kg group, lowered surface skin temperatures were observed in 4 litters (3 of which were litters of dams in which bad development of mammary glands and bad nest building were observed), and an external wound on the tail was seen in one case.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

For the males, no adverse effects of reproductive capability were seen even at 1000 mg/kg/day (highest) dose.

As to females, seven of the nine females lost their pups in the 1000 mg/kg group. Numbers of corpora lutea and implantation scars were decreased in 1000 mg/kg group.

With regard to pups, decreased body weights of both sexes at birth were seen in the 100 mg/kg or more groups. Decreased numbers of live pups born, live birth index and viability index, and decreased body weights of both sexes on day 4 after birth were seen in the 1000 mg/kg/group.

[Summary on the reproduction]
1) In all doses, there was no significant difference from control group in the following items.
* Estrous cases before mating (14 days)
* Copulation index, fertility index.
* Length of gestation (days)

2) In 30 to 300 mg/kg/day doses, there was no significant difference from control group in the following items.
*Number of corpora lutea
*Number of implantation scars
*Implantation index.:
  
3) In 1000 mg/kg/day group,the significant lower values were seen in corpora lutea and implantation scars. 7 out of 9 females lost their pups.

[Summary on pups production]
1) Number of pups born: It was 100% for all groups except for a pup was not born in 30 mg/kg/day. This was not considered due to the adverse effect of divinylbenzene dose.
2) In 30 to 100 mg/kg/day, no significant difference from control group in the birth index.
3) In 30 to 300 mg/kg/day groups, no significant difference from control groups was seen on the following items.
* Number of the pups born and the delivery index
* Number of the live pups born, the live birth index and the sex ratio at
  birth.
*Number of live pups on day 4 of lactation and viability index.
4) In more than 100 mg/kg/day groups, the significant low body weight of pups at birth was seen.
5) At 1000 mg/kg/day group,the significant lower value of the number of pups born and the lower tendency of the delivery index. The significant lower values were observed in number of the live pups born and the live birth index. The lower tendency in body weight on day 4 lactation was seen also.
6) No external abnormality due to the dose of DVB was observed in all the groups

Applicant's summary and conclusion

Conclusions:
For the males, no adverse effects of reproductive capability were seen even at 1000 mg/kg/day (highest) dose. As to females, seven of the nine females lost their pups in the 1000 mg/kg group. Numbers of corpora lutea and implantation scars were decreased in 1000 mg/kg group. The NOAELs for reproductive performance are considered to be 1000 mg/kg/day for males, and 300 mg/kg/day for females.

With regard to pups, decreased body weights of both sexes at birth were seen in the 100 mg/kg or more groups. Decreased numbers of live pups born, live birth index and viability index, and decreased body weights of both sexes on day 4 after birth were seen in the 1000 mg/kg/group. The NOAELs for pup development is considered to be 30 mg/kg/day.
Executive summary:

This study was conducted in accordance with an OECD Combined Repeated Dose and Reproductive/Development Toxicity Screening Test. For the males, no adverse effects of reproductive capability were seen even at 1000 mg/kg/day (highest) dose. As to females, seven of the nine females lost their pups in the 1000 mg/kg group. Numbers of corpora lutea and implantation scars were decreased in 1000 mg/kg group. The NOAELs for reproductive performance are considered to be 1000 mg/kg/day for males, and 300 mg/kg/day for females. With regard to pups, decreased body weights of both sexes at birth were seen in the 100 mg/kg or more groups. Decreased numbers of live pups born, live birth index and viability index, and decreased body weights of both sexes on day 4 after birth were seen in the 1000 mg/kg/group. The NOAELs for pup development is considered to be 30 mg/kg/day.