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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from authoritative database

Data source

Reference
Reference Type:
other: J-check
Title:
One-Generation Reproduction Toxicity Test of the test chemical in Rats
Author:
National Institute of Technology and Evaluation
Year:
2018
Bibliographic source:
J-check

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 415 (One-generation reproductive toxicit study)
Deviations:
yes
Principles of method if other than guideline:
Developmental toxicity test of test chemical was carried out using Sprague-Dawley rats.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-Naphthol
Cas Number:
135-19-3
Molecular formula:
C10H8O
IUPAC Name:
2-Naphthol
Test material form:
solid: flakes
Details on test material:
- Name of test material: 2-naphthol
- Molecular formula: C10H8O
- Molecular weight: 144.172 g/mol
- Substance type: Organic
- Physical state: Solid: grey flakes
- Impurities (identity and concentrations): purity: 99.6%, gray white flake containing 0.3% of test chemical as an impurity

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Remarks:
Sprague-Dawley (SD) (Crj: CD (SD) IGS, SPF)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Tsukuba breeding center, Charles River Japan Co., Ltd.
- Age at study initiation: 4 weeks (males) and 7 weeks (females)
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: housed individually in a metallic wire mesh floor cage in a breeding room
- Use of restrainers for preventing ingestion (if dermal): yes/no: No data available
- Diet (e.g. ad libitum): Solid feed (CE-2, Japan CLEA Co., Ltd.) ad libitum
- Water (e.g. ad libitum): tap water, provided ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1 ° C
- Humidity (%): 50 to 65%
- Air changes (per hr): about 15 times / hour
- Photoperiod (hrs dark / hrs light): lighting for 12 hours (lights up at 7-19 o'clock)

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 w/v%
Remarks on MMAD:
No data available
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in 0.5 w/v % aqueous solution of carboxymethylcellulose sodium (CMC Na) with the help of magnetic stirrer to achieve the dosing concentrations of 0, 10, 40 mg/kg or 160 mg/kg

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5 w/v % CMC Na
- Concentration in vehicle: 0, 10, 40 mg/kg or 160 mg/kg
- Amount of vehicle (if gavage): 5ml/kg
- Lot/batch no. (if required): No data available
- Purity:No data available
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data available
Details on mating procedure:
The mating procedure was performed with 1:1 (one male to one female) mating in the study. One female was placed with the same male until the pregnancy occurred or three weeks elapsed. Each morning the females were examined for presence of sperm or vaginal plug. Day 0 of pregnancy was identified as the day a vaginal plug or sperm were found.
Duration of treatment / exposure:
14 weeks (98 days of administration)
Frequency of treatment:
Daily
Duration of test:
No data available
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
40 mg/kg bw/day
Dose / conc.:
160 mg/kg bw/day
No. of animals per sex per dose:
0 (vehicle control)= 25male and 25female
10mg/kg= 25male and 25female
40mg/kg= 25male and 25female
160 mg/kg= 25male and 25female
Control animals:
yes, concurrent vehicle
Details on study design:
Males were dosed 10 weeks before mating, during mating and they were necropsied one week after the end of the mating period. Females received the test chemical 2 weeks before mating, during 3 weeks of mating and pregnancy till the 21st day of lactation (overall 98 days). Dams were allowed to deliver pups naturally after mating and they were necropsied with their offspring on the 22nd day of lactation. Both males and females continued to be administered until the day before necropsy. During the entire administration period the general condition of the parent animals, weight gain and changes in food intake were recorded. Parameters of reproductive ability, including gonadal function, oestrus cycle, mating behaviour, conception, parturition and lactation and weaning of pups were observed.

- Dose selection rationale: The dose for this study was set based on the results of a preliminary study using Sprague-Dawley rats (same strain used in the main study). The test chemical of the test chemical was suspended in CMC Na, and 0, 40, 120 or 360 mg / kg was repeatedly administered orally to 2 males and 5 females in each group from 2 weeks before mating. Females were necropsied onthe 14th day of gestation, and males were necropsied on the 28th day after the first administration. Severe toxic effects including deaths were observed in the highest (360 mg/kg) dose group, therefore the doses used in the main study were reduced by ratio 4. Thus, the low dose was set as 10 mg/kg, medium dose as 40 mg/kg and the high dose as 160 mg/kg. As a result, although it is feared that some deaths may occur, it is expected that toxic changes are observed relatively frequently. The dose was set at a high dose of mg / kg, and was reduced by a common ratio of 4. The medium dose was 40 mg / kg and the low dose was 10 mg / kg.

- Rationale for animal assignment (if not random): The animals were divided into groups according to body weight stratified random sampling method, and 25 males and females were placed in each group.
- Fasting period before blood sampling for clinical biochemistry: No data
- Other: No data

Examinations

Maternal examinations:
Maternal animals were observed for mortality, general condition, change in weight, food intake and oestrus cyclicity. Animals were subject to gross pathological examinations to determine the presence or absence of gross abnormalities of major organs in the thoracic-abdominal area including lung, stomach, liver, kidney, spleen, bone marrow of femur, pituitary glands, and reproductive organs such as ovary, uterus (horns and cervix) and vagina.
Ovaries and uterine content:
Oestrus cycle, number of implantations and gestation length were determined.
Fetal examinations:
The general conditions, the number of live pups, litter count and pup body weights and pup survival index were measured/calculated.
Statistics:
Fisher's direct probability test was carried out on frequency of type of sex cycle, mating rate, conception rate, morphological abnormality frequency of babiesAccording to histopathological examination findings, the Mann-Whitney U test shows the grade-separated data, and the total value of the positive grade is obtained by Fisher's direct probability one-sided test between the control group and each test substance administration group Significant difference test was carried out. For the other data, we tested the uniformity of variance of each group by Bartlett method , with the value obtained for each individual, or the average value for each litter as one sample . If the variance is uniform, a one-way analysis of variance was performed, and when significance was observed between the groups, multiple comparisons were performed according to the Dunnett method . On the other hand, Kruskal-Wallis rank test is performed when the variance is 0 in any group and when the variance is not uniform, and if significance is observed between the groups.
Indices:
Copulations and Fertility indexes were determined in males and females. Survival index for pups was calculated.
Historical control data:
No data available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Males: Transient salivation after drug administration was observed in all treated groups. In the 10 mg/kg group, salivation was observed only in one case at the beginning of treatment. However, the number and frequency of cases increased in the 40 mg/kg group and in the 160 mg kg dose group almost all animal showed salvation during entire administration period. In addition, decrease in locomotor activity, ptosis or eyelid closure were observed at 40 mg/kg or more, and lacrimation and the nasal discharge group in the 160 mg/kg administration group. In the 160 mg/kg group, reduction in size of feces and the excretion of black loose feces were also temporarily observed. Clinical signs observed independently of the dose of the test substance were hair loss in 10 and 160 mg/kg groups and scab formation in the 10 and 40 mg/kg groups. In addition to these findings, damage to the upper incisors was temporarily observed in one case in the 40 mg/kg dose group, and one case with abnormality in the upper jaw position was observed from 91 days after administration until necropsy.

Females: Decrease in locomotor activity and salivation were observed transiently after administration in the administration group of 40 mg/kg or more. These clinical signs were observed only at the initial stage of administration in the 40 mg/kg administration group but were observed over the entire period including the lactation period in the 160 mg/kg administration group, and the frequency also increased depending on the dose. In addition, nasal discharge was observed in the 40 mg/kg group, and lean and prone positions were also observed in the 160 mg/kg group at the initial stage of administration. In addition, in one case of the 160 mg/kg administration group in which all the offspring died on the day of delivery, abnormal respiratory noise and hair contamination on the vulva were observed on that day, and then hair contamination extended to the abdomen. Hair loss was observed in a few cases in all administration groups, independently from doses, and tears in the perineal area were observed in 1 case of the 40 mg/kg administration group.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
In males, one animal in the 160 mg / kg dose group died on the 60th day of administration due to incorect operation during administration.
In females, there were no deaths or moribundity.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In males, there were no significant differences in either body weight between the control and the test substance administrated groups. In females, no significant difference was found between the control and any of the treated groups regarding body weight before mating and during pregnancy and only slight decrease in the postpartum body weight were seen in the high-dose (160 mg/kg) group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males no change in food consumption were observed and in females no change in food consumption before and during mating, but significantly lower values than control group, after pregnancy and during the nursing stage
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Decreased locomotor activity was noted initially after the test item administration in the 40 mg/kg group and in all administration periods of the 160 mg/kg group in maternal animals. As the incidence of decreased locomotor activity increased by the dose they were regarded as treatment-related clinical signs.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
The swelling of liver was observed in the only male from 160 mg/kg group which, died due to mishandling during drug administration. At necropsy, a thickened area of the anterior gastric mucosa was observed in one case in both 40 and 160 mg/kg administration groups. Enlargement of liver was observed in two cases in the 160 mg/kg dose group. Degenerative lesion of the kidneys and unilateral dilatation of renal pelvis were seen in all groups including control thus the changes appeared independently of the doses used. In the 40 mg /kg group very slight to slight degenerative lesions were observed in the liver, spleen, bone marrow
and lung. There were no dose-dependent changes in male and female reproductive organs. Very slight and slight degeneration of spermatids at the 14th stage of spermatogenesis in the seminiferous tubule of testis were observed in all treated groups. However, this impaired spermatogenesis was detected at low frequency of occurrence and severity.Pathological examination of the reproductive organs revealed no treatment-related and dose-dependent changes in male and female gonads at any dose level. There was no significant difference in the number of implantations in the uterus between the control and any of the administrated groups. Squamous hyperplasia of the anterior gastric mucosa was observed in the 40 and 160 mg/kg groups were the only significant and treatment-related findings in males during necropsy, although in females, no effect was observed in any tissues.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
Stomach: Squamous hyperplasia of the anterior gastric mucosal squamous epithelium was observed in in males of 160 mg/kg group. In females no abnormalities were observed.

Pituitary: No abnormalities were observed.

Testis and epididymis: In cases where fertility was not confirmed, one case each in the 40 mg / kg and 160 mg / kg administration groups was limited to seminiferous tubules in the 14th stage of the spermatogenesis cycle, and spermatocytes were selected. Mild degeneration was observed, and cell debris was observed in the lumen of the epididymis in the 40 mg / kg group. No abnormalities were observed in the other cases.

Degeneration of seminiferous cells localized to seminiferous tubules in the 14th stage was observed in a few cases in each test substance administration group, and in many of the cases epididymal cell debris were observed in the lumen in cases where fertility was confirmed. However, for these findings, no significant difference was found between the control group and each test substance administration group. Seminal vesicles: No abnormalities were observed in the seminal vesicles.

Prostate ventral lobe: Lymphocyte infiltration was observed in the stroma of the control group and the 160 mg / kg administration group, and in cases where the degree of infiltration was strong, neutrophils into the epithelium and lumen were found

Coagulation gland: No abnormalities were observed

Abnormal organs at autopsy: In the control group, basophilic tubules were found in the cortex and cylinders in the cortex and medulla were observed in the kidneys where unilateral reduction was observed. Eosinophilic body was also observed in the kidney where unilateral dilatation of the renal pelvis was observed.

In the 40 mg / kg group, no abnormality was observed in the darkened liver. A case of abnormal separation with liver lightening, bilateral miniaturization and lightening of kidney, swelling with lightening of spleen, swelling of adrenal gland, and lightening of brain, lung and femur bone marrow In the liver, localized necrosis and single cell necrosis of hepatocytes and extramedullary hematopoies is were observed in the liver, but significant enhancement of granulocyte production was also observed in the spleen in addition to extramedullary hematopoiesis of red blood cells in the spleen. Enhancement of bulb production was observed. In addition, in this example, the lungs contained a large number of foam cells, diffuse neutrophilic infiltration of arteries and capillaries, and basophilic tubules were observed in the renal cortex, but abnormal in the brain and adrenal glands. There were no other findings in the kidneys of cases where dilation of the renal pelvis was observed.

In the 160 mg / kg group, no abnormalities were observed in the liver of the cases where swelling was observed. In the kidney where dilation was observed in the unilateral renal pelvis, basophilic tubules were also observed in the cortex.

Females: In addition to localized embolism with mineral deposits in the blood vessels of one uterine horn muscle in the control group, infertility was observed in the control group and the 160 mg / kg administration group, as well as in the other administration groups No abnormalities were observed in the pituitary, stomach, ovaries, uterus, cervix and fistula of the cases. In addition, no abnormality was observed in the liver of the 160 mg / kg administration group in which a diaphragmatic nodule was observed at autopsy, but there were hair follicles in the skin of the 10 mg / kg and 160 mg / kg administration groups in which hair loss was observed. A decrease in focalness was observed.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Loose feces and hair loss were also observed in both males and females in various dose groups, although these effects did not show dose-dependence in their frequency or severity.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The number of implanantions did not differ significantly from control.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
The number of live pups was significantly lower in the 160 mg/kg group at lactation day 4 and 7 as compared to controls and the viability index at lactation day 4 was significantly lower in the same group as compared to controls.
Early or late resorptions:
not specified
Dead fetuses:
not specified
Description (incidence and severity):
After parturition, in mg/Kg group, all offspring died at the time of labor confirmation.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No change in the birth rate and gestational period were seen between the control group and each test substance administration group.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No changes in the number of pregnant females, number of pregnant females with live newborns and birth rate were detected between the control group and each test substance administration group.
Other effects:
no effects observed
Description (incidence and severity):
No abnormalities during labour were observed, and no change of the birth rate was seen. As for parturition, abnormalities were observed in one case each in the 40 mg / kg and 160 mg / kg groups. These included bloody contamination of the perianthral coat or bleeding from the stoma.

In one animal in the 160 mg / kg group, nursing behavior such as collecting infants on the first day after birth was not recognized, and all birth children died the next day. No abnormalities were observed in other animals.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
changes in number of pregnant
changes in pregnancy duration
early or late resorptions
effects on pregnancy duration
maternal abnormalities
necropsy findings
pre and post implantation loss
total litter losses by resorption
other: Labour condition, birth rate

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males, no significant difference was found between the control group and each test substance a dministration group at any time.

As for females, in the 10 mg / kg and 160 mg / kg groups, body weight at 21 days of birth was significantly lower (p <0.05) compared to the control group. There was no significant difference between the 40 mg / kg group and the control group at any time.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The number of live pups was significantly reduced at 160 mg/kg at PND 4 and 7. In the high-dose group the number of live pups were reduced but not significantly.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no effect of test chemical administration on pups sex ratio.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
The litter size was decreased in 160 mg/kg/day group.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
In 160 mg/kg/day group the postnatal survival declined significantly as compared to control.
External malformations:
no effects observed
Description (incidence and severity):
No test item-related external malformations were seen at any dose group. Morphological changes including malformations and mutations were observed in the offspring of each 1-2 litters of each group including the control group. Changes in the external surface include small eyes in one belly of the control group, mild subcutaneous hematomas in one belly of the 10 mg / kg dose
group, and pitting after peeling of the skin in one belly of the 160 mg / kg dose group was observed. Of these, only the small eyes in the control group were classified as malformations.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No test item-related skeletal malformations were seen at any dose level.
Visceral malformations:
no effects observed
Description (incidence and severity):
No test item-related visceral malformations were seen at any dose level. Dilation of the renal pelvis, which is one of the visceral mutations, was observed in each belly of the 10 mg / kg and 40 mg / kg groups. As a result of calculating the total number of the offsprings showing these morphological changes in the offsprings after autopsy and comparing the frequency, no significant difference was found between the control group and each test substance administration group.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
The number of live pups was significantly lower in the 160 mg/kg group at lactation day 4 and 7 as compared to controls and the viability index at lactation day 4 was significantly lower in the same group as compared to controls.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
other: No growth inhibition

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
160 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
No test item-related abnormalities were found during gestation and parturition of maternal animals, but the prenatal exposure to the test chemical produced adverse effects on neonatal growth and viability at the concentration of 160 mg/kg or more which occurred in the absence of severe maternal toxicity. Hence, the NOAEL for reproductive and developmental toxicity is considered as 160 mg/kg for both parental animals because of the lack of test chemical-induced changes in sexual function and fertility. The reproductive and developmental NOAEL for F1 generation was concluded as 40 mg/kg due to the decreased neonatal survival rate, when male and female SD rats were orally treated with the test chemical for 98 days.
Executive summary:

A one-generation reproductive toxicity study was performed to investigate the toxic nature of the test chemical when it is administered to male and female Sprague-Dawley rats. Groups of 25 animals/dose/sex were treated with 0, 10, 40, 160 mg/kg of test chemical by gavage for 14 weeks (98 days). Males were dosed 10 weeks before mating, during mating and they were necropsied one week after the end of the mating period. Females received the test chemical 2 weeks before mating, during 3 weeks of mating and pregnancy till the 21stday of lactation (overall 98 days). Dams were allowed to deliver pups naturally after mating and they were necropsied with their offspring on the 22ndday of lactation. Both males and females continued to be administered until the day before necropsy. General conditions, clinical signs, changes in body weight gain and food intake of parental animals were observed during the administration period. The effects of the test substance on male and female reproductive performance including gonadal function, oestrus cycle, mating behaviour, conception, parturition and lactation and weaning were followed. The early neonatal survival and the growth of offspring were also recorded. Transient salvation and decreased locomotor activity were found in all administration groups in both sexes, although their incidence increased by the dose thus, they were regarded as treatment-related clinical signs. Nasal discharge, lean and prone position, ptosis (eyelid closure) and lacrimation were also observed in both males and females in various dose groups, although these effects did not show dose-dependence in their frequency or severity. In males, there were no significant differences in either body weight or food consumption between the control and the test substance administrated groups. In females, no significant difference was found between the control and any of the treated groups regarding body weight before mating and during pregnancy and only slight decrease in the postpartum body weight were seen in the high-dose (160 mg/kg) group. In females, the food consumption dropped significantly in lactation day 4-21, in the 160 mg/kg dose group. No significant changes were seen in oestrus cyclicity, conception rate and the number of days required from the start of cohabitation to copulation in any of the treatment females in comparison with the controls. As for parturition, single cases of abnormalities were observed in the two highest dose groups and in one case within the 160 mg/kg dose group, all offspring died due to complicated labour. However, no abnormalities were found in the delivery status of the other animals, and there were no significant differences in the birth rate and gestation period between the control and any of the treated groups. The reproductive performance described by the copulation and fertility indexes of both males and females were comparable with controls. Degeneration of seminiferous cells localized to seminiferous tubules in the 14th stage was observed in a few cases in the 40 and 160 mg/Kg dosed animals and in many of the cases epididymal cell debris were observed in the lumen in cases where fertility was confirmed. However, for these findings, no significant difference was found between the control group and each test substance administration group. Similarly, no differences between control and treated groups were seen regarding the rearing conditions. The lack of nursing behavioural was noticed in one female of 160 mg/kg group which led to the death of the litter, but no abnormal nursing behaviour were observed in other animals. Pathological examination of the reproductive organs revealed no treatment-related and dose-dependent changes in male and female gonads at any dose level. There was no significant difference in the number of implantations in the uterus between the control and any of the administrated groups. Squamous hyperplasia of the anterior gastric mucosa was observed in the 40 and 160 mg/kg groups were the only significant and treatment-related findings in males during necropsy, although in females, no effect was observed in any tissues.In the F1 generation, skin abnormalities (peeling off the skin) were seen at the high-dose (160 mg/kg) group from Day 0. In addition, the number of live pups was significantly lower in the 160 mg/kg group at lactation day 4 and 7 as compared to controls and the viability index at lactation day 4 was significantly lower than those of control in the same group. Moreover, no significant differences were found between the control and any of the treated groups regarding external and visceral malformations and the total number of the offspring showing morphological abnormalities was comparable with controls in frequency. As the birth weight was equal to that in the control group, it was considered that there was no growth suppression in utero either. In addition, no neonatal growth failure was observed among pups. Thus, it was postulated that the significant increase in death during the early neonatal period either caused by a direct effect of the test chemical on offspring viability or by the slightly suppressed maternal lactating activity due to the mild hypnotic effect of the test chemical. In summary, no abnormality was found infertility, reproductive function and parturition of parental animals, but the prenatal exposure to the test chemical produced adverse effects on neonatal growth and viability at the concentration of 160 mg/kg or more which occurred in the absence of severe maternal toxicity. Hence, the NOAEL for reproductive toxicity is considered as 160 mg/kg for both males and females because of the lack of the test chemical-induced changes in sexual function and fertility when male and female SD rats were orally treated with the test chemical for 98 days. The developmental NOAEL for maternal and F1 generation was concluded as 40 mg/kg due to the decreased neonatal survival rate.