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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:
1996-01-12 to 1997-07-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study, carried out by Bozo Research Center Inc. (Japan)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline for testing of Chemicals, Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crj: CD(SD) SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: Approximately for male and female (P) 8 wks; (F1) x wks
- Weight at study initiation: (P) Males: 302-326 g; Females: 206-232 g; (F1) Males: x-x g; Females: x-x g
- Housing: Individually bred in a metal cage (W 190 x D 350 x H 170 mm: Lead Engineering Co.,); except hybridization period from day 17 to nursing day 4; During hybridization period from evening to following morning, one male and one female were accommodated in a metal net cage (W 266 x D 266 x H 200 mm: RIKO Electric Industry Co.,)
- Diet (e.g. ad libitum): solid food (NMF: Oriental Yeast Co.,) ad libitum
- Water (e.g. ad libitum): drinking water (Fujimi Water Association: Automatic water supply) ad libitum. During breeding period, drinking water was supplied by water bottle
- Acclimation period: approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5±23.5°C
- Humidity (%): 50±20%
- Air changes (per hr): ventilation frequency 10 to 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day (7 a.m. to 7 p.m.)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The necessary quantity of the test substance was weighed for each administration, and prepared at necessary temperature by dissolving in water for injection. By the way, the test substance was not purity converted, and indicated as original weight.
The administration dose was determined for 0.5 mL/100g

Individual administration solutions were calculated based on, for males, the weight on the day of measurement, and for females, during mating period weight on each measuring day, and during gestation period weight on gestational day 0, 7, 14 and 21, and during nursing day 0.

For the administration period, the forced administration was performed in accordance with the Guideline (OECD Guideline for testing of Chemicals, Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test) using a metal Magen Sonde, once a day between 9 a.m. to 3 p.m.
Details on mating procedure:
After the administration during 14 days, one male and one female as one pair of a same group were lived together all-night. The cohabitation period was maximum 14 days until the confirmation of copulation. Copulation was verified everyday by checking copulatory plug or presence of spermatozoa in vaginal smear, once confirmed female as acopulated animal, the day was indicated as gestational day 0. Because the male supposed to cross (100 mg/kg dos group: 2003) have died, a female of 100 mg/kg dose group (No. 2103) was bred with another male (No. 2002) of the same group who was verified for copulation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before administration start for both sexes and at final week of administration to males, twice by gas chromatography for each concentration used, consequently ratio to the indicated value was respectively within range of 99.0 ~ 105.0 %
Duration of treatment / exposure:
Males: 49 days from 14 days before mating to the day before autopsy
Females: 41 to 45 day from 14 days before mating, mating period (5 days maximum), gestation period and nursing day 4 until the day before autopsy
Frequency of treatment:
Once daily
Details on study schedule:
Test period:
January 12, 1996 to July 31, 1997

Receipt dato of animals:
February 19, 1996

Administration start date:
February 26, 1996 (male, female)

Mating (Hybridization) start date:
March 11, 1996

Delivery start date:
April, 3 1996

Female animals autopsy start date:
April 7, 1996

Male animals autopsy date:
April 15, 1996

Animal test completion date:
April 15, 1996
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
12 per sex per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
A 2 weeks preliminary test of oral administration using rats was conducted to determine the concentration range (Test No.: 0 -417, dose: 0, 125, 250, 500 and 1000 mg/kg). For the administration of 1000 mg/kg, there was no apparent effect of the test substance in performance status, weight, amount of food consumption, haematological data and blood chemical laboratory data. Therefore, the high-dose in the test was defined to 1000 mg/kg, then subtracting by a common ratio 3, medium and lower doses were respectively set to 300 and 100 mg/kg.
Positive control:
none

Examinations

Parental animals: Observations and examinations:
1) Males (P)

(1) Observation of Performance Status
For the performance status, symptom of toxicity and behavioural problems were observed everyday 3 times a day, before and right after and 2 hours after the administration. But, for day-off, observation was twice before and after the administration, on the day of the autopsy once before the autopsy.

(2) Measurement of Body Weight
Weight was measured between 8 a.m. to 12:30 p.m. on administration starting day (before administration on day 1), on administration day 4, 8, 11, 15, 22, 29, 36, 43 and on autopsy day (day 50 after the administration).

(3) Measurement of Food consumption
The amount of food consumption was measured between 8 a.m. to 12:30 p.m. of the same day with body weight except on autopsy day, calculated from difference with amount of food consumption of previous day.




2) Female (P)

(1) Observation of General Appearance
For the general appearance, symptom of toxicity and behavioural problems were observed everyday 3 times a day, before and right after the administration and 2 hours after. But, for day-off, observation was twice before and after the administration, on the day of the autopsy once before the autopsy.

(2) Observation of Estrus Cycle
For the observation of estrus cycle, vaginal smear was collected and observed at the microscope everyday from the start of the administration until the confirmation of copulation. During the administration before mating, the vaginal smear images (picture, statues, statue) were classified by proestrus, estrus, postestrus and anestrus, and then estrus manifestation count and days (estrus cycle) from estrus to the following estrus were checked. Besides, the vaginal smear on the day before the administration was excluded from calculation of estrus cycle. During mating period, the presence of spermatozoa was checked.

(3) Measurement of Body Weight
Weight was measured between 8 a.m. to 12:30 p.m. on administration day (before administration on day 1), on administration day 4, 8, 11 and 15, for during gestatation period on gestational day 0, 7, 14 and 21, and during nursing period on nursing day 0 and 4.

(4) Measurement of Food Consumption
The amount of food consumption was checked between 8 a.m. to 12:30 p.m. on the same day with weight measured, for during gestation period on gestational day 1, 7, 14 and 21, and for during nursing period on nursing day 1 and 4, calculated from difference with amount of food consumption of previous day.

(5) Observation of Delivery and Nursing
The dams were left for natural delivery to observe the presence of anomaly delivery. The completion of delivery was checked twice everyday from gestational day 21 to 25, when the delivery has completed before 10 a.m. and the day was indicated as nursing day 0. For one case (No. 1106) in the control group, as delivery was not observed before 10 a.m. on the pregnant day 25 after confirmation of copulation, the autopsy was performed after exsanguination under lethal ether anaesthesia to verify formation of pregnancy, because implantation was not observed in utero, it was considered to be sterile.
Dams confirmed for the completion of delivery were left to nurse pups born, and continued observation of nursing everyday until the nursing day 4.

Oestrous cyclicity (parental animals):
(2) Observation of Estrus Cycle
For the observation of estrus cycle, vaginal smear was collected and observed at the microscope everyday from the start of the administration until the confirmation of copulation. During the administration before mating, the vaginal smear images (picture, statues, statue) were classified by proestrus, estrus, postestrus and anestrus, and then estrus manifestation count and days (estrus cycle) from estrus to the following estrus were checked. Besides, the vaginal smear on the day before the administration was excluded from calculation of estrus cycle. During mating period, the presence of spermatozoa was checked.
Sperm parameters (parental animals):
During mating period, the presence of spermatozoa was checked; no more data
Litter observations:
4) Neonate (F1)

(1) Observation of Neonatal Pups
At nursing day 0, survivals and stillborn children were counted and observed for the presence of body surface anomaly. Survival pups were checked for the sex, and after weighed, and all they were left nursed by dams. The stillborn children were fixed and conserved in 10% formalin conditioned with phosphate buffer solution (1/15M, pH 7.1 -7.4) except those who showed marked deterioration of postmortem change.

(2) Observation and Measurement of Nursing Pups
Nursing pups were observed once a day for their life and death. The weight was measured in nursing day 0 (birth day) and day 4. On the nursing day 4, after all nursing pups were exsanguinated under lethal ether anaesthesia, by autopsy the presence of anomaly of internal organs was observed.
Postmortem examinations (parental animals):
1) Males (P)

(4) Haematological Examination
All animals were abstained from food for overnight (16 hours) at slaughtering autopsy of the following day after the completion of the administration, then, underwent an abdominal surgery by ether aneasthesia, then collected blood from abdominal aorta.

a) With the use of the collected blood from abdominal aorta in a blood bottle (SB-41: Toa Medical Electronic Co.,), supplemented with anticoagulant (EDTA-2K), the following measurements were performed:

Coulter 8 -parameter Automated Hematology Analyzer (Nikkaki Co.,) used: RBC count [Electric Resistance Change Detection Method], Hemoglobin [Cyanmethemoglobin Method], Hematocrit [Mean RBC volume (µ^3) X RBC count (10^4/mm^3)/10^3], Mean RBC volume [Electric Resistance Change Detection Method], Mean RBC heoglobin [Hemoglobin (g/dl) X 10/RBC count (10^6/mm^3)], Mean RBC hemoglobin concentration (Hemoglobin (g/dl) X 10^2/Hematocrit (%)], Platelet count and WBC count [Electric Resistance Change Detection Method].
Reticulocyte count [Brecher Method], WBC percentate [May-Giemsa staining at microscopy].

b) Take the collected blood in a test tube with 3.8% sodium citrate, after centrifuged (3000 rpm, 10 minutes), the following measurements were performed with the obtained plasma.

Coagulation Analyzer ACL100 (Instrumentation Laboratory) used: Prothrombin time and Activated partial thromboplastin time [Clot Method], Fibrinogen [Thromboplastin Method].

(5) Blood Chemical Test
The following test was performed using collected blood from abdominal aorta at the same time with blood collection for heamtological examination.

a) Take the collected blood in a test tube, after centrifuged (3000 rpm, 10 minutes), the following measurements were performed with the obtained plasma:

Automatic analyuer [Monarch (Instrumentation Laboratory)] used: AIP [Bessey-Lowry Method], Total cholesterol [CEH-COD-POD Method], Trygliceride [GK-GPO-POD Method], Phospholipid t [PLD-ChOD-POD Method], Total bilirubin [Azobilirubin method], Glucose [Hexokinase-GLDH method], Urea nitrogen [Urease-GLDH method], Creatinine [Jaffe method], Sodium and Potassium and Chlorine [In selective electrode method], Calcium [OCPC method], Phosphor [Molybdate method], Total protein [Bluret method].
Ectrophoresis system [CLINISCAN 2 (Helena Kenkyujyo Co.,) used: Protein fraction [Cellulose acetate menbrane elctrophoresis].
A/G ratio [Calculated from protein fraction ratio].

b) The collected blood from abdominal aorta in a tube containing heparin was centrifuged (3000 rpm, 10 minutes), the following measurements were performed with the obtained plasma.

Automatic analyuer [Monarch (Instrumentation Laboratory)] used: GOT [UV-rate assay], LDH [UV-rate method].
Coulter 8 -parameter Automated Hematology Analyzer (Nikkaki Co.,) used: GPT [UV-rate assay].

(6) Autopsy
After all animals were weighed on the following day of the last administration (50 days after the start of administration), and after collection of blood under ether anaesthesia, internal organs and tissues were observed macroscopically by autopsy. Furthermore, brain, heart, lung with bronchial, liver, spleen, hypophysis, thyroid with parathyroid, adrenal, thymus, kidney, testicle and epididymis were collected, internal organs and tissues were fixed in 10% formalin conditioned with phosphate buffer solution (1/15M, pH 7.1 -7.4) except testicle and epididymis which were conditioned in Bouin´s solution. Also, died animal (100 mg/kg dose group: 2003) was treated similarly to the survivals.

(7) Measurement of Organ Weight
For organs, heart, thymus, liver, kidney, spleen, testicle and epididymis were weighed, then, relative organ weight was calculated from final body weight.

(8) Histopathological Examination
For all animals, heat, thymus, liver, kidney, spleen, testicle, epididymis and gross abnormal regio at autopsy were paraffin embedded. For all animals of the control group, of the highest-dose group and of dead animal, a segment was made, then observed at the microscope after eosin staining.


2) Female (P)

(6) Autopsy
At nursing day 4, autopsy was performed for all dams after exsanguination under lethal ether anesthesia to observe internal organs and tissues macroscopically, also, number of corpus luteum and implantations traces were counted as well. Furthermore, brain, heart, lung with bronchial, liver, spleen, hypophysis, thyroid with parathyroid, adrenal, thymus, kidney, ovary, uterine, vagina and gross abnormal regio were collected and fixed in 10 formalin conditioned with phophate buffer solution (1/15M, pH 7.1 -7.4). The sterile animal (control group: 1106) was treated similarly.

(7) Measurement of Organ Weight
For organs, heart, thymus, liver, kidney, spleen and ovary were weighed, then, relative organ weight was calculated from final body weight.

(8) Histopathological Examination
For all animals, heat, thymus, liver, kidney, spleen and ovary were paraffin embedded. For all animals of the control group and of the high-dose group, a segment was prepared, then, observed at the microscope after hematoxylin-eosin staining. In addition, for the case of a liver suspected for the effect of the test substance, segment was prepared for whole group and searched similarly. Furthermore, for 2 examples (1109, 1110) of the control group and 2 examples (4102, 4103) of 1000 mg/kg dose group, the liver was searched by PAS staining.
Postmortem examinations (offspring):
4) Neonate (F1)

(1) Observation of Neonatal Pups
At nursing day 0, survivals and stillborn children were counted and observed for the presence of body surface anomaly. Survival pups were checked for the sex, and after weighed, and all they were left nursed by dams. The stillborn children were fixed and conserved in 10% formalin conditioned with phosphate buffer solution (1/15M, pH 7.1 -7.4) except those who showed marked deterioration of postmortem change.

(2) Observation and Measurement of Nursing Pups
Nursing pups were observed once a day for their life and death. The weight was measured in nursing day 0 (birth day) and day 4. On the nursing day 4, after all nursing pups were exsanguinated under lethal ether anaesthesia, by autopsy the presence of anomaly of internal organs was observed.
Statistics:
Significant level was determined for 5 and 1%. For the children, the average of one litter was defined as one unit.

Multiple Test:
The test of distribution uniformity of each group was performed by Barlett´s test. Therefore, when the distribution is uniform, the analysis of variance by One Way Layout was performed, and when there is a significant difference between groups, paired comparison test was performed using the Dunett Test when identical number of samples in each group, and the Scheffé test when different number of samples between groups. When the dispersion was not uniform, the Kruskal-Wallis test was performed, and if significant, for the difference of average of the ranks, the Dunett Test was performed for identical number of samples in each group, and the Scheffé test for different number of samples between groups
Body weight, amount of food consumption, estrus manifestation count, estrus cycle, cohabitation days, gestation period, hematological examination, blood chemical test, organ weight, corpa lutea count, implantation traces, number of children born, implantation rate, sex ratio, delivery rate, birth rate and survival rate in neonatal pups


Chi-Square Test:
Copulation rate, pregnancy rate and childbirth rate


Calculation of Various Data:
Copulation rate (%) = (No. of copulation verified animals/No. of mating animals) X 100
Conception rate (%) = (No. of pregnant animals/No. of copulation verified animals) X 100
Childbirth rate (%) = ( No. of dams with live birth/No. of pregnant dams) X 100
Pregnancy period (day) = Nursing day 0 (delivery verified date) - gestation date 0
Sex ratio = Males/(males + females)
Neonatal survival rate (%) = (No. of survivals on nursing day 4/No. of lives on nursing day 0) X 100
Delivery rate (%) = (Total childbirth pups/No. of implantation traces) X 100
Birth rate (%) = (No. of survivals on nursing day =/Total childbirth pups) X 100
Implantation rate (%) = (No. of implantation traces count/No. of corpus luteum) X 100
Reproductive indices:
see Table 1
Offspring viability indices:
see Table 1

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males, a decrease in RBC was seen in the 100 mg/kg bw/day dose group, which was not throught otbe treatment related. All other parameters were similar in treatment groups compared to controls.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males, urea nitrogen was lower in the 3000 mg/kg bw/day dose group compared with controls, and alpha-1-glubulin was lower in the top dose group. Both were unrelated to treatment.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The disappearance of lipid droplets in hepatocytes and an increase in glycogen and liver weight were seen in females at 1000 mg/kg bw/day, likely to be related to pregnancy. No changes in clinical chemistry were noted hence the toxicological significance of such changes is considered to be quite low.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

I. Repeat Dose Toxicity

1. Effect to Males

1)Performance Status
In 7 cases ( No. 4004, 4006, 4007, 4009, 4010, 4011, 4012) of the 1000 mg/kg dose group, salivation was seen 29 days after the administration. This salivation was expressed right after the administration, and continued until at the completion of administration period. This salivation disappeared 1 hours after the administration.
And, 1 case of the 100 mg/kg dose group (No. 2003) had died on the administration day 17. For this case, there was no change in performance status, weights and amount of food consumption until the day before death, though lung became dark reddish, histological alveolus edema and humor retention with pale red foamy substance was seen, the cause of death was estimated to be an incorrect administration. Also, in other case in the control group (No. 1008), a maxillary tooth crack was observed on administration day 32.

2) Weight
The weight in the test substance group has changed during administration period similarly to the control group, and the significant difference in weight on each measuring day and in weight gain during the administration period was not seen between the control groups.

3) Food Consumption
The amount of food consumption in the test substance administration showed approximately similar value with the control group through the administration period, and the significant difference in amount of food consumption on each measuring day was not seen between the control groups.

4) Hematological Findings
Though a significantly low value of RBC was seen in the 100 mg/kg group, there was no association with the administered dose. Other examination parameters in the test substance dose group showed approximately the same values with control group, and the significant difference was not seen between the control group and the test substance dose group.

5) Blood Chemical Test
A significant low value of urea nitrogen was seen in the 300 mg/kg dose group, but there was no association with the administration dose. Also, a significant low value of a alpha 1 -globulin fraction was observed was seen in 1000 mg/kg dose group, a slight increase of albumin ratio and A/G ratio in protein fraction were observed in this dose group. Other parameters in the test substance dose group have shown approximately the same value with the conrol group, significant difference was not seen between the control group and the test substance dose group.

6) Autopsy Findings
A diffusion of dark reddish spots in lung was observed in one case (No. 4008) of the 1000 mg/kg test group. In other cases, there was no macroscopic abnormaly.

7) Organ Weights
A significant high value of the relative weight of spleen was observed in the 300 mg/kg dose group, but, there was no association with the administered dose. Other, in the absolute and relative weights of the measured organs and tissues, i.e. thymus, heart, kidney, testis and epididymis, the significant difference was not seen between the control group and the test substance dose group.

8) Histopathological Findings
The change related to the test substance administration was not recognized for the histopathological examination carried out for 1000 mg/kg dose group. But, the minor fat development of hepatic cell in liver lobe peripheral increased extramedullary hematopoiesis in spleen were observed in all cases in the control group and the 1000 mg/kg dose group.
In addition, eosinophilic body was found in 2 cases (1002, 1006) in the control group, and in 3 cases (4001, 4003, 4006) in the 100 mg/kg dose group, furthermore, restricted atrophy of seminiferous tubule in testis and restricted infiltration of inflammatory in lung were observed in one case (No. 4008) in the group of 1000 mg/kg dose group, but as these changes were either minor or slight, they were considered contingent from their manifestation.


2. Effect to Females

1) General Appearance
There were no death during before and during mating period, gestational period and through to nursing period.
Change in general appearance before and during mating period was not observed neither in the control group nor in the test substance dose group.
During gestational period, at the observation right after the administration, salivation was seen continuously until 10 days after the gestation, in 5 cases (No. 4101, 4104, 4107, 4110, 4112) of the 1000 mg/kg dose group. This salivation disappeared similarly to males 1 hour after the administration. No other general appearance was observed.
Also during nursing period, similarly to the gestational period, salivation was seen continuously from postpartum to nursing day 3 in 5 cases (No. 4104, 4105, 4107, 4110, 4112) of the 1000 mg/kg dose group. No other performance status was observed.

2) Body Weights
The weight in the test substance group has changed similarly to the control group through mating period, gestation period and to nursing period, and the significant difference in weight on each measuring day and in weight gain during each period was not seen between the control group and the test substance dose group.

3) Food Consumption
The amount of food consumption in the group of the test substance dose group has shown through mating period, gestation period and to nursing period, approximately the similar values to the control group, and the significant difference in amount of food consumption on each measuring day was not seen between the control group an the test substance dose group.

4) Autopsy Findings
An adhesion of adipose tissues on spleen, liver, stomach and on periphery to these organs was seen in one case (No. 4108) in 1000 mg/kg dose group. In other case, there was no macroscopic anomaly.

5) Organ Weight
In the group of 1000 mg/kg dose group, a significant high value of absolute and relative weight in liver and the rise of absolute weight in liver and the rise of absolute weight in bilateral kidney and a significant high value of its relative weight were seen. Though a significant difference was not seen in the group, the absolute and relative value of thymus have showed a decline trend. Furthermore, in the absolute and relative weight of the measured organs and tissues, i.e. thymus, heart, spleen and ovary, the significant difference was not seen between control group and the test substance dose group.
For the 300 mg/kg dose group, in the absolute and relative weight of the measured organs and tissues, the significant difference was not seen between the control group and the test substance dose group.

6) Histopathological Findings
In the 1000 mg/kg dose group, a change in liver was seen, which could be associated to the test substance administration. In other words, the increase of glycogen accumulated in hepatic cells was seen slightly in 8 cases in the control group, in 9 cases in the 100 mg/kg dose group, and in 8 cases in 300 mg/kg dose group, but in all cases of 1000 mg/kg dose group, it was minor or slightly, a significant increase of number of manifestation was recognized between the control group and the 1000 mg/kg dose group. Also, minor fat development of hepatic cell in liver lobe peripheral was seen in 10 cases in the control group, in 6 cases in the 100 mg /kg dose group and 6 cases ub tge 300 mg/kg dose group, as opposed to no observation in the 1000 mg/kg dose group, a significant increase of number of manifestation was recognized between the control group and the 1000 mg/kg dose group. Therefore, in the 1000 mg/kg dose group, it was considered that fat development in hepatic cells disappears, and amount of glycogen accumulated in hepatic cells increases. Besides, similarly to males, a slight increased extramedullary hematopoiesis in spleen was seen in all cases of the control group and of 1000 mg/kg dose group, and slight capsule fibrosing was seen in one case (No.4108) in the 1000 mg/kg dose group which had also adhesion in spleen at autopsy, although the significant difference in manifestation of these findings was not seen between the control group and the test substance dose group, it was considered contingent due to their manifestation.

II. Reproductive Toxicity

1. Effect to Parent Animals

1) Estrus Cycle
In the test substance dose group, the estrus cycle i.e. estrus manifestation count and days from estrus to following estrus was approximately similar to the control group, and the significant difference was not seen between the control group and the test substance dose group.
For each individual, in one case (No. 4108) in the 1000 mg/kg dose group, an anestrus was observed continuously, though no anomaly was seen in estrus cycle for other cases. Besides, for one case (No. 4108) in 1000 mg/kg dose group, which showed anomaly of the estrus cycle, copulation was confirmed at mating day 5, no anomaly was recognized for impregnation capability. Also, as there was no other cases of estrus cycle anomaly in the same group, it was considered as a contingent change independent from the test substance administration.

2) Copulation Rate and Impregnation Rate
In either of the control group and of the test substance dose group, copulation was formed within 5 days after cohabitation, the copulation rate in each group was 100%. There was no significant difference in average days required for formation of copulation between the control group and each test substance group. The formation of copulation was observed in all pairs except in one pair (No. 1006 X 1106) in the control group, and the pregnancy rate in the test substance dose group was 100%.

3) Number of Neonatal Pups Birth Females, Gestation Period, and, Nursing Conditions
In one case (No. 1106) in the control group, delivery was not observed even at pregnancy day 25, as a result of autopsy it was steril. Other cases in the control group and in the test substance administration group, completion of delivery was confirmed by pregnancy day 23, but there was no significant difference in pregnancy period between the control group and the test substance dose group. In addition, anomaly of delivery was not observed, and birth rate was 100% for each group. For the nursing conditions, there was no anomaly in nursing behaviour such as nesting, pup approach and lactation in every case in the control group and in the test substance group.

4) Corpus Luteum Count, Implantation Traces, Implantation Rate and Number of Neonatal Pups Births
The number of corpus leuteum, implantation traces and implantation rate showed approximately the same value with the control group, there was no significant difference between the control group and the test substance group. Besides, stillbirth were seen only in 3 dams (No. 2101, 2107, 2109) of the 100 mg/kg dose group, and in 2 dams (No. 3103, 3111), number of children born in the test substance dose group showed approximately the same value with the control group, thus there was no significant difference in delivery rate between the control group and the test substance dose group.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 3 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

2. Effect to Neonatal Pups

1) Sex Ratio, Body Surface Anomaly, Survivals and Existence Rate
There was not significant difference in sex ratio between the control group and the test substance dose group. For the observation of the body surface of neonatal pups, no significant difference was observed between the control group and the test substance dose group. Number of survivals and birth rate in the test substance group showed approximately the similar value with the control group, no significant difference was observed between the control group and the test substance dose group. Additionally, neonatal pups died during up to 4 days after birth were seen in 2 cases in 2 dams (No. 2106, 2111) of the 100 mg/kg dose group, and in 6 cases in 3 dams (No. 3103, 3108, 3109) of the group, though no significant difference was observed in survival rate on the day 4 after birth between the control group and the test substance group.

2) Body Weights
The weights on the birth and on the day 4 after birth in the test substance dose group showed approximately similar value with the control group, no significant difference was observed between the control group and the test substance dose group.

3) Autopsy Findings
At the autopsy on the day 4 after birth, anomaly was not seen either in the control group or in the test substance dose group.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see Remark
Remarks on result:
other: Sex ratio, body surface anomaly, survival, existence rate, body weights, autopsy findings

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Reproductive data

Dose level(mg/kg/day)   0     100      300      1000
No. of pairs mated     12      12       12        12
No.of pregnant females 11      12       12        12
No.of pregnant females 11      12       12        12
with pups alive
Gestation index      100.0   100.0     100.0     100.0
Gestation length in days (Mean ± SD)        
                   22.5±0.5 22.7±0.5 22.7±0.5  22.3±0.5
Number of corpora lutea (Mean ± SD)      
                      205      230       234       238
                 (18.6±2.4)(19.2±1.5)(19.5±3.1)(19.8±2.6)
No. of implantations (Mean ± SD)        
                      195      204       209       215
                 (17.7±2.3)(17.0±1.9)(17.4±2.1)(17.9±1.4)
Implantation index    95.1     88.7     89.3      90.3 
Day0 of lactation No. of stillborn (Mean ± SD)
                       0        3         2        0.0
                   (0.0±0.0)(0.3±0.5) (0.2±0.4) (0.0±0.0)
No. of  live born (Mean ± SD) 
                      184      187       195       201  
                  (16.7±2.0)(15.6±2.2)(16.3±2.6)(16.8±1.9)
Delivery index        94.4     93.1      94.3      93.5
Live birth index      100      98.4      99.0      100
Sex ratio(Mean±SD)    0.51     0.48      0.55      0.52
Day 4 of lactation Number of pups alive (Mean±SD)
                      184       185       189      197
                  (16.7±2.0)(15.4±2.2)(15.8±2.2)(16.4±1.8)
Viability index       100      98.9       96.9     98.0
Body weight of live pups (grams) (Mean±SD)
on day 0  
Males               6.9±0.8   7.2±0.7   7.1±0.7  6.71±0.5
Females             6.6±0.7   6.7±0.7   6.6±0.7   6.3±0.5
on day 4

Applicant's summary and conclusion

Conclusions:
The reproductive NOAEL, under the test conditions, is 1000 mg/kg/day.

Executive summary:

As part of OECD investigation project of toxicity in regard to existing chemical safety review, a combined repeat dose toxicity and reproduction toxicity test of 3 -Methyl-1,5 -Pentanediol by oral administration in rat was performed. In other words, dose 0 (control group), 100 and 300 and 1000 mg/kg of the substance was orally administered to SD rat (Crj=CD); for males, during 49 days from 17 days before mating start through to mating period until the day before autopsy, for females, 41 to 45 days from 14 days before mating start through to mating period, gestation period after copulation formation and delivery until nursing day 3. Then, the effect of general toxicity in male and female animals by repeat administration was searched as well as the effect to reproductive ability and formation and growth of following generation such as the gonad function, copulation behavior, impregnation and delivery and nursing.

Repeat Dose Toxicity:

1. Effect to Males (P)

In the group of 1000 mg/kg administration, for observation of performance status, approximately in a half of cases, salivation was manifested 29 days after the administration. This salivation was expressed right after the administration, and changed to disappear in 1 hour after the administration. Also, in the blood chemical test, a decrease of alpha 1 -globulin fraction ratio and a slight increase of albumin and A/G ratios in protein fraction were observed. The effect by the administration of the test substance was not observed in weight, amount of food consumption, hematological and in pathological findings. In 300 and 100 mg/kg dose groups, effect caused by the administration of the test substance was not observed in any of performance status, body weight, food consumption, hematological, blood chemical and pathological findings.

2. Effect to Females (P)

In the group of 1000 mg/kg, for the observation of performance status, similarly in males, salivation right after the administration was seen in about half of cases at after gestational day 10. Furthermore, in the pathological findings, an increase of glycogen and liver weight involving disappearance of lipid droplet of hepatic cell was observed. In the group of 300 and 1000 mg/kg administration groups, the effect caused by the administration of the test substance was not observed in any of performance status, body weight, amount of food consumption and pathological findings.

Consequently, under the test conditions, the general toxic No Observed Adverse Effect Level (NOAEL) is estimated at 300 mg/kg/day for both males and females.

Reproduction Toxicity

1. Effect to Reproductive Toxicity (P)

In the group of 1000 mg/kg dose group, the effect of the administration of the test substance was not observed in any of female estrus cycle and estrus frequency, sexual copulation and impregnation rates, female gestation period, delivery and nursing status, furthermore, nor in corpus luteum count, implantation traces and implantation rate, number of neonatal pups born and delivering rate.

2. Effect to Neonatal Pups (F1)

Also in the group of 1000 mg/kg dose group, the effect of the administration of the test substance was not observed in number of survival at nursing 0 day, number of stillborn infants and birth rate and sex ratio, there was no neonate with anomaly in body surface. In addition, the effect of the administration of the test substance was not seen in survival rate and body weight on the day 4 after birth, and anomaly was not noted for autopsy as well.

Consequently, the reproductive toxic No Observed Adverse Effect Level (NOAEL), under the test conditions, is estimated at 1000 mg/kg/day for both parental animals and neonatal pups.