6-tert-butyl-2,4-xylenol

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not specified

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: “The order for specifying the items for the study on new chemical substances, and the investigation on the designated chemical substances. Article 4: Test Facility,” Kankiken No. 233, Eisei No. 38, 63 Kikyoku No. 823 (November 18, 1988).

Deviations:

no

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

6-tert-butyl-2,4-xylenol

EC Number:

217-533-1

EC Name:

6-tert-butyl-2,4-xylenol

Cas Number:

1879-09-0

Molecular formula:

C12H18O

IUPAC Name:

2-tert-butyl-4,6-dimethylphenol

Test material form:

liquid: viscous

Details on test material:

Name of the test substance: 6-tert-butyl-2, 4-xylenol
CAS No.: 1879-09-0
Code No.: B0903
Purity: 98.5%
Storage conditions: room temperature, in the dark
Storage place: archive for test substance, in BioSafety Research Center
Chemical name: 6-tert-butyl-2, 4-xylenol
Molecular formula: (CH3)3CC6H2(CH3)2OH
Molecular weight: 178.27
Description: liquid
Color: very pale yellow, transparent
Solidification point: 21.5°C
Solubility: water-insoluble
Specific gravity (d20/20): 0.9612
Refractive index (n20/D): 1.5186
Precaution for handling: Appropriate eye protective device, protective gloves and protective mask were worn during handling. Hand washing and sufficient gargle were performed after handling.
Storage of test substance and Disposal of the residue: After the end of administration, about 2 g of test substance was stored in BioSafety Research Center, and the remainder was discarded.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals: Seventy males and 70 females of Crj:CD(SD) strain rats (SPF) were purchased from Japan Charles River Co., Ltd., (Atsugi-shi, Kanagawa) at 8-weeks of age on September 20, 1993, quarantined and acclimatized to the test environment for 7 days. 48 males and 48 females which showed no abnormalities in general condition during the acclimatization period were allocated to each group after 8 days of preliminary housing period at 10-weeks of age.
At the end of grouping, the males weighed 339-400 g and the females weighed 226-265 g.

Animal husbandry: The animals were housed in an animal room (W 5.7 x D 10.0 x H 2.5 m, 142.5m3) kept by barrier system under the target environmental conditions at 22-26°C of temperature, 45-65 % of relative humidity with 15 times per hour of ventilation frequency, and 12 hour lighting of 150-300 lux (lighting at 7: 00 am and turn off at 7:00 pm).
Animals were housed individually in a cage with aluminum front and stainless steel mesh-floor (W 15.8 x D 23.8 x H 16.0 cm, Space: 6017 cm3). Cages were set on a water-flush breeding instrument (W 691.0 x D 79.0 x H 195.0 cm) supplied by Tokyo Giken Service Co. Ltd. (Fuchu-shi, Tokyo). However, during the mating period, males were housed in cages with aluminum front and stainless steel mesh-floor (W 36.8 x D 25.0 x H 16.0 cm, Space: 14720 cm3). Dam after 18 days of gestation was housed in a cage with aluminum front and stainless steel mesh-floor (W 36.8 x D 25.0 x H 16.0 cm) with a lactation tray and nesting materials (Alpha-dry) until day 4 of lactation. The cages were exchanged once every other week, and food suppliers were exchanged once a week.
The food used was NMF solid food (treated with radiation sterilization) supplied by Oriental Yeast Co., Ltd. (Chuo-ku, Tokyo). The food was available ad libitum. The analysis of contaminant in the food used in this study was conducted in Japan Food Research Laboratories (Shibuya-ku, Tokyo) on the request by Oriental Yeast Co., Ltd.
The animals were allowed free access to drinking water of tap water. The analyses of tap water were performed 4 times in a year in Examination Center of Life Science of Shizuoka (Hamamatsu-shi, Shizuoka). The food, water and nesting material supplied did not contain any contaminants which were considered to have affected the results of the study.
There were no environmental deviations which might have affected the reliability of the study data during the duration of housing period.

Test groups: Animals were stratified by the body weights and allocated to each test group by randomization method on October 5, 1993.
Animals were identified by ear-punch and attaching animal identification card with animal identification number (animal ID-No.) to cages. Before grouping, animals were identified by tentative animal numbers.
The remained animals were euthanized by carbon dioxide.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Exact amount of test substance was dissolved into corn oil (Nacalai Tesque, Kyoto-Shi, Kyoto) to make the specified concentrations of 1.2, 6 and 30 mg/ml. After the preparation, the preparations were stored in a refrigerator until use. As shown in Reference data 5, the test substance preparation was conducted at the frequency of once a week or more, and the preparations were used within 7 days after preparation. In the case of 1.2 mg/ml, the preparation was confirmed to be stable at least for 8 days after preparation.

Details on mating procedure:

After 14 days of observation period for estrus cycles before mating, female animals were placed in cages together with male animal of the same group in one-to-one base for 2 weeks as the longest period. In the next morning, the presence of sperms in the vaginal smears was determined as the established copulation and as 0 day of gestation. The observation of estrus cycles was conducted until the day of copulation. The number of days between each estrus stage were estimated to be the number of days of estrus cycle, and mean estrus cycle durations were calculated.
From the results of mating, the copulation index [(number of animals of copulation/number of animals placed together) x 100] was calculated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis of homogeneity and concentration of the dosing preparation was conducted on the sample taken randomly from batches of each group prepared at the start of administration. The mean values of the concentration in each preparation were within 99.3-104 %, and the coefficient of variation was lower than 0.8 % indicating that the preparations were properly adjusted.

Duration of treatment / exposure:

Male animals were administered for 45 days consecutively. Female animals were administered for 41-48 days. Infertile female animal was administered for 44 days until the day before necropsy.

Frequency of treatment:

The test substance was administered by gavage once a day (7 days/week) using a stomach tube.

Details on study schedule:

Male animals were administered for 45 days consecutively from 14 days before mating, during 14 days of mating period and up to 17 days after the end of mating period. Female animals were administered from 14 days before mating, during mating period (until the day of copulation confirmation, 14 days at the longest), gestation period after copulation and up to day 3 of lactation after delivery (41-48 days). Infertile female animal was administered for 44 days until the day before necropsy (day 25 of gestation).

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 6, 30, 150 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

12 animals of each sex per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Reason for selection of the species: The use of rat is indicated by OECD Guideline. The species was selected considering the stability in estrus cycle, reproduction and genetic.
Reasons for the selection of dose levels: Dose levels were selected by considering the results of “Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Preliminary Study in Rats” (study number 2265) conducted previously. In this preliminary study, dose levels of 0, 75, 150, 300 and 600 mg/kg were administered to males and females for 14 consecutive days, and all animals treated with 600 mg/kg died. Suppression in body weight gain was seen in male groups treated with 300 mg/kg or more and in females treated with 600 mg/kg. Decrease in food intake was seen in male groups treated with 150 mg/kg or more, and females treated with 300 mg/kg or more.
Among the organ weight at necropsy, both absolute and relative weights of liver increased in male groups treated with 75 mg/kg or more, and in female groups treated with 150 mg/kg or more.
At necropsy, enlargement of liver was seen in males treated with 150 or 300 mg/kg and females treated with 300 mg/kg.
From the above results and taking into account of the longer administration period in the main study, 150 mg/kg was selected as the highest dose level. The lower dose levels of 30 and 6 mg/kg were selected by dividing the highest dose level by common geometric ratio of 5.

Positive control:

Positive control not utilised in this study.

Examinations

Parental animals: Observations and examinations:

Mating: After 14 days of observation period for estrus cycles before mating, female animals were placed in cages together with male animal of the same group in one-to-one base for 2 weeks as the longest period. In the next morning, the presence of sperms in the vaginal smears was determined as the established copulation and as 0 day of gestation. The observation of estrus cycles was conducted until the day of copulation.
From the results of mating, the copulation index [(number of animals of copulation/number of animals placed together) x 100] was calculated.

Observation on the natural delivery and lactation period: The observations of delivery were done from 9:00 am to 10:00 am during days 20-25 of gestation. By confirmation of the completion of delivery during this observation time, the day was recorded as day 0 of lactation. In the case of delivery after 10:00 am, the next day was recorded as day 0 of lactation.
In addition, gestation period (number of days between day 0 of gestation and day 0 of lactation), fertility index [(number of pregnant animals/number of copulated animals) x 100], gestation index [(number of females with live birth/number of pregnant female) x 100], implantation index [(number of implantation sites/number of corpora lutea of pregnancy) x 100], delivery index [(number of birth/number of implantation sites) x 100], live birth index [(number of live birth/number of birth) x 100] were calculated.
Female which showed no delivery until 9:00 am on day 25 of gestation was necropsied, and female without implantation site was judged to be infertile.
At the natural delivery, the status of delivery was observed, and the condition of lactation was observed until day 4 of lactation. At the necropsy on day 4 of lactation, numbers of corpora lutea of pregnancy and implantation sites were counted.

Oestrous cyclicity (parental animals):

The number of days between each estrus stage were estimated to be the number of days of estrus cycle, and mean estrus cycle durations were calculated.

Sperm parameters (parental animals):

Not specified

Litter observations:

After parturition, number of birth (live birth + still birth) were examined, and sex was identified and sex ratio (males/females) was calculated. Presence of external anomaly was also examined. In addition, body weight of each litter was weighed separately by sex on day 0 and 4 of lactation and mean body weight of each sex was calculated.
Furthermore, the viability index on day 4 [(number of live neonates on day 4 of lactation/number of live birth) x 100] were calculated.

Postmortem examinations (parental animals):

Male animals
Necropsy and organ weight: Macroscopic observations of organs and tissues were performed on the animals euthanized by exsanguinations after blood sampling. Thymus, liver, kidney, testis and epididymides were weighed, and the ratio of organ weight to body weight (relative weight) was calculated. In addition, thymus, liver, kidney, brain, heart, spleen, adrenals, seminal vesicles, prostate, pituitary and the tissues with abnormal findings (lung, mass in the abdominal cavity) of all animals were fixed in 10% neutral buffered formaldehyde solution. The testis and the epididymides were fixed in Bouin solution.
Histopathology: Paraffin sections from the fixed organs and tissues indicated below were prepared by Histo Science Laboratory Co., Ltd., (Oume-shi, Tokyo) and routinely stained with hematoxylin-eosin. In addition, kidney was stained with PAS reaction. Microscopic observations were conducted in BioSafety Research Center.
Fertile male: Brain, thymus, heart, liver, kidney, spleen, adrenals, testis of all animals of the control group and 150 mg/kg group and the tissues with abnormal findings in all groups. Thymus, liver, kidney and adrenals of all animals of 6 and 30 mg/kg groups.
Infertile male: Brain, thymus, heart, liver, kidney, spleen, adrenals, testis, epididymides, seminal vesicles, prostate and pituitary.

Female animals
Necropsy and organ weight: The animals necropsied are shown below and the presence of abnormalities in the organs and tissues was observed.
Dead animal: Animals were necropsied immediately after found dead. Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord were fixed in 10% neutral buffered formaldehyde solution.
Female with natural delivery: Animals were euthanized by exsanguination under ether anesthesia on day 4 of lactation, and main organs were observed macroscopically. And then, thymus, liver, kidney and ovaries were weighed, and the ratio of organ weight to body weight (relative weight) was calculated. In addition, thymus, liver, kidney, ovaries, brain, heart, spleen, adrenals, pituitary and the tissues with abnormal findings of all animals were fixed in 10% neutral buffered formaldehyde solution.
Female without natural delivery: On day 25 of gestation, animals were euthanized by exsanguinations under ether anesthesia, and macroscopic observations of main organs were performed. Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord were fixed in 10% neutral buffered formaldehyde solution. Animal with no implantation sites was judged to be infertile.
Female whose pups all died: On the day or the next day when the death or cannibalism of pups were found, parental females were euthanized by exsanguinations under ether anesthesia, and macroscopic observations of main organs were performed. Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord were fixed in 10% neutral buffered formaldehyde solution. The numbers of corpora lutea of pregnancy and implantation sites were examined at the necropsy.
Histopathology: Paraffin sections from the fixed organs and tissues indicated below were prepared by Histo Science Laboratory Co., Ltd., (Oume-shi, Tokyo) and routinely stained with hematoxylin-eosin. In addition, kidney were stained with PAS reaction. Microscopic observations were conducted in BioSafety Research Center.
Dead animal: Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord.
Female with natural delivery: Brain, thymus, heart, liver, kidney, spleen, adrenals, ovaries of all animals of the control group and 150 mg/kg group, and the tissues with abnormal findings of all animals of all groups. Thymus, liver, kidney and adrenals of all animals of 6 mg/kg group and 30 mg/kg group.
Infertile female: Brain, thymus, heart, liver, kidney, spleen, adrenals, vagina, ovaries and pituitary.
Female whose pups all died: Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord.

Postmortem examinations (offspring):

On day 4 of lactation, all neonates were sacrificed and main organs were macroscopically observed. Neonates died during lactation period were also necropsied and main organs were macroscopically observed.

Statistics:

Data of this study were recorded using a computer system, and the results of the study were statistically analyzed using the following methods.
The mean value per dam was used as one sample for the results of neonates during lactation period. Levels of significance were two steps of * : P < 0.05 and ** : P < 0.01.

Multiple comparison analysis was applied for body weight, food intake, number of corpora lutea of pregnancy, implantation site, number of birth, number of still birth, sex ratio, mean value of estrus cycle duration, gestation period, implantation index, delivery index, live birth index, incidence of external anomaly, viability index on day 4 of neonates, organ weight, ratio of organ weight to body weight (relative organ weight), hematology and blood chemistry.
First, the homogeneity was assessed by Bartlett test. If homogeneity was obtained, one way analysis of variance was applied. If variance was significant and the numbers of samples were equal among groups, Dunnett multiple comparison was applied. In the case of unequal numbers of samples, the significances of difference between the control group and each treated group were assessed by Scheffé multiple comparison.
When the data showed non-homogeneity by Bartlett test, the data were analyzed by Kruskal-Wallis rank sum test. Dunnett rank test was applied if significance was obtained and numbers of sample were equal, and in the case of unequal numbers of samples, Scheffé rank test was applied for the analysis of significant difference between the control group and each treated group.
X2 test was applied for gestation index, copulation index and fertility index.
Fisher’s direct probability method was applied for the enlargement of liver in macroscopic findings and the centrilobular hypertrophy of hepatocytes in microscopic examination.

Reproductive indices:

Multiple comparison analysis was applied for number of corpora lutea of pregnancy, implantation site, number of birth, number of still birth, sex ratio, mean value of estrus cycle duration, gestation period, implantation index, delivery index, live birth index, incidence of external anomaly.

Offspring viability indices:

Multiple comparison analysis was applied for viability index on day 4 of neonates

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Details on results (P0)

Effect on parental animals
Copulation and fertility: Copulation was established in all animals in all groups.
Fertilization was successful in all of test substance administered groups, but was unsuccessful in one pair of the control group. In the estrus cycle observation, pseudopregnancy (continuous diestrus smear image) was not observed, and estrus cycles of 4-5 day were seen in all groups and no inter-group differences were seen in the mean duration of estrus cycles.

Delivery and lactation: The mean durations of gestation were within the range of 22.5-22.9 days in each group, and no prolongation of gestation was observed. One animal (animal No. 2305) in 150 mg/kg group delivered 9 neonates and died during delivery. At necropsy, uterus was examined and 6 residual fetuses were found in the uterus. In addition, the number of implantation sites in 150 mg/kg group decreased compared to the control group. The number of females whose pups all died during lactation period was 4 (animal No. 2204, 2303, 2309, 2312). Viability index on day 4 were slightly lower both in male and female of 150 mg/kg group. However, no statistically significant differences were noted. No abnormal delivery was seen in the control group, 6 mg/kg group or 30 mg/kg group. No inter-group differences were observed in the number of corpora lutea, number of implantation sites, sex ratio, gestation index, implantation index, delivery index, live birth index and viability index on day 4 of neonate.

Effect levels (P0)

1

Key result

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Detailed in results

Body weight and weight changes:

no effects observed

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:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Effect on neonates
External examination: No external abnormalities were seen in all groups.
Body weight: No inter group differences were seen both in male and female in the body weights until day 4 of lactation.
Necropsy: In the necropsy of dead neonates until day 4 of lactation, thymic remnant in the neck was sporadically seen in test substance administered group.
In the necropsy of live neonates sacrificed on 4 day of lactation, thymic remnant in the neck, white patch/zone in liver, pale and pelvic dilatation in kidney were sporadically seen in the control group, 6 mg/kg group and 30 mg/kg group.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

gross pathology

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

This report was translated from the original Japanese report. The translated copy is appended below for reference.

Applicant's summary and conclusion

Conclusions:

From the results, no effect in male fertility by the administration of 6-tert-butyl-2, 4-xylenol was seen at the dose level of 150 mg/kg/day. Therefore, no observed effect level is estimated to be 150 mg/kg/day.
Concerning to the effect in female reproduction and development and growth of neonate, numbers of dams whose pups all died increased in 150 mg/kg group. Therefore, no observed effect level is estimated to be 30 mg/kg/day.

Executive summary:

In order to evaluate the toxicological nature of existing chemical substances, the repeated dose toxicity as well as the effect on reproduction and development were studied by the administration of 6-tert-butyl-2, 4-xylenol at the dose levels of 0 (vehicle control), 6, 30 and 150 mg/kg/day to rats for the period from 14 days before mating, during mating period, gestation period and to day 3 of lactation.

 

The general toxicological effect as well as the effect on reproduction and development were studied following the OECD Guidelines for “Testing of Chemicals; Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Screening Test, Extended Steering Group Document No. 3” (March 22, 1990).

The conduct of the study was designed to fulfill the standard described in “The order for specifying the items for the study on new chemical substances, and the investigation on the designated chemical substances. Article 4: Test Facility,” Kankiken No. 233, Eisei No. 38, 63 Kikyoku No. 823 (November 18, 1988).

 

No effect due to the test substance administration was observed in the copulation capability, fertility or estrus cycle observation. In the observation at parturition, one female of 150 mg/kg group died during delivery. In addition, there were 3 females whose pups all died in this group. The viability index on day 4 was inclined to show low value, and it is suggested that the test substance has the possibility to induce disorder in parturition or lactation function. However, no effect due to the test substance administration was seen during gestation period or delivery duration. In the external examination of neonates, no external anomalies were observed, and the body weight of neonates increased normally until day 4 of lactation. Abnormal findings considered to be due to the test substance administration were not seen in the rate of stillbirth and death rate of pups, or on the necropsy on day 4 of lactation.

From the above results, no effect on male fertility were observed by the administration of 150 mg/kg/day. Therefore, no observed effective level is estimated to be 150 mg/kg/day. The effect on fertility of female and development and growth of neonates were observed by the administration of 150 mg/kg/day. Therefore, no observed effective level is estimated to be 30 mg/kg/day.