2,5-bis-isocyanatomethyl-bicyclo[2.2.1]heptane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A EOGRTS is currenlty being performed. Submission date of this study is mentioned in the study record.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

In an oral prenatal toxicity study, performed according to OECD guidline 414 and in accordance with GLP principles, in rat the maternal NOAEL was 150 mg/kg bw/day and the developmental NOAEL was at least 500 mg/kg bw/day, the highest dose tested.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March 2021 - 30 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study was performed to assess the potential maternal toxicity and embryo fetal toxicity of NBDI.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

CAS No.: 74091-64-8
Molecular weight: 206.24
Stability: Stable until the expiration date under the specified conditions.
Description: Colorless and transparent liquid, pungent odor.
Solubility: insoluble in water; soluble in oil
Storage conditions: Store in a tight container at cool place (acceptable range: 1 to 15°C), protect from light, and keep dehumidification.

Species:

rat

Strain:

other: Crl:CD(SD) [SPF]

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: Females: 9 weeks
- Weight at study initiation: The body weights of the pregnant females assigned to the test group on Day 0 of gestation ranged from 223.2 to 310.1 g.
- Fasting period before study: No
- Housing: Animals were individually housed in steel mesh cages
hung on a water flushing breeding rack. The cages and feeders were changed once every two weeks and once every week, respectively. During the gestation period animals were provided with an aluminum tray and bedding material.
- Diet: ad libitum, pellet diet CRF-1 (sterilized by irradiation, Oriental Yeast)
- Water: ad libitum
- Contaminants: Levels of contaminants in diet and water where within acceptable limits proposed by the Japan Experimental Animal Feed Association.
- Acclimation period: 15 (in the study report, the term from Day -15 to -9 is called the quarantine period, and the term from Day -8 to -1 the acclimation period. During the quarantine and acclimation periods, the animals were observed once daily and weighed three times)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C (actual values: 22.5 to 23.8°C)
- Humidity (%): 35 to 70%RH (actual values: 49 to 62%RH)
- Air changes (per hr): 12 times air changes or more per hour
- Photoperiod (hrs dark / hrs light): 12 hours (light on: 7:00, light off: 19:00)

IN-LIFE DATES: From: 15 March 2021 To: 30 June 2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: For the dose levels of 50, 150 and 500 mg/kg bw/day, the dosing formulations of 12.5, 37.5 and 125 mg/mL were prepared respectively.
At first, 125 mg/mL solution was prepared. A required amount of the test substance was accurately weighed into a measuring cylinder and mixed with an adequate amount of the vehicle. Then the vehicle was added up to the final volume. Dosing formulations of 37.5 and 12.5 mg/mL were prepared by diluting 125 mg/mL solution. The prepared dosing formulations were aliquoted for daily use, and stored in the refrigerator until use [actual temperature: 4.1 to 7.1°C, duration of storage: from March 11, 2021 to April 6, 2021 (from the first day of preparation of dosing formulations to the final day of dosing)]. The dosing formulations were used within a period to assure their stability.

VEHICLE
- Justification for use and choice of vehicle: not reported
- Concentration in vehicle: 12.5, 37.5 and 125 mg/mL for the dose levels of 50, 150 and 500 mg/kg bw/day, respectively.
- Amount of vehicle: 0.4 mL per 100 g body weight
- Lot/batch no.: SKP4098
- Purity: not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

STABILITY/HOMOGENEITY OF THE DOSING FORMULATIONS
The 10 and 200 mg/mL dosing formulations were analyzed just after preparation, after 4 and 8 days of storage in a tight light-resistant container under refrigeration (acceptable range: 1 to 9°C) and subsequent each 6 hours of storage in a tight light-resistant container at room temperature (acceptable range: 1 to 30°C). Remaining (ratio of each mean measured concentration after storage to those just after preparation) and relative standard deviation (RSD) were calculated from the measured concentration. When the remaining are within 100.0±10.0%, the test substance formulations would be judged to be stable. When the RSD are 5.0% or less, the test substance formulations would be judged to be homogeneous.

CONCENTRATION/HOMOGENEITY OF THE DOSING FORMULATIONS
Concentrations of NBDI in the dosing formulations were examined at the first and last preparations by the method established previously [Validation of Determination method of NBDI in corn oil: Exp. No. J353 (575-015)]. Whether each formulation have been appropriately prepared or not was judged according to the following criteria.

- Vehicle (for the control animals): No interference peak should be detected at the retention time of NBDI on the chromatogram. If the peak is detected, the peak area should be less than 10.0% of that of the standard solution for calibration curve (nominal concentration: 0.1 mg/mL).
- Test substance-containing formulation: Concentration: The ratio to the nominal concentration is within 100.0±10.0%. Homogeneity: The relative standard deviation is 5.0% or less.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight until evidence of mating was confirmed
- Estrus cycles of all animals were examined for at least 7 days prior to mating, and those animals in a suitable condition for mating were subjected to the mating process.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: presence of sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 5 of gestation until day 19 of gestation.

Frequency of treatment:

Once daily

Duration of test:

-evidence of mating: GD 0
-assignment of pregnant females to study groups: GD 0
-exposure to the test substance: GD 5 - GD 19
-euthanasia and sample collection: GD 20

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

(only vehicle was administered)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

0 mg/kg bw/day: 23 pregnant females
50 mg/kg bw/day: 23 pregnant females
150 mg/kg bw/day: 23 pregnant females
500 mg/kg bw/day: 21 pregnant females (In the uterine examination on Day 20 of gestation, one animal in the 500 mg/kg/day group was judged to be aborted during the gestation period. Therefore, the numbers of corpora lutea and implantations of this animal were adopted. Other data of this animal, such as findings of clinical signs and gross findings, values of body weight, food consumption and hormones were excluded from the calculation. In addition, organ weights measurement and histopathological examination of thyroid glands were not conducted.)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a preliminary dose range finding study (BSRC study number J354 (575-016)) aiming at the investigation of prenatal developmental toxicity of NBDI in rats (dose levels: 0, 100, 500, 700 mg/kg bw/day, N=5), 1/5 animal died in the 700 mg/kg bw/day group. In the 500 and 700 mg/kg bw/day groups, lower food consumption and decreased body weights were observed in the early stage of administration period, and suppression of body weight gains were noted. In the macroscopic examination, red patches on the forestomach and thickening of forestomach were observed in almost all animals in the 500 and 700 mg/kg bw/day groups. In addition, thickening of forestomach was observed in one non-pregnant animal in the 100 mg/kg bw/day group.
From these results, 500 mg/kg bw/day was set as the high dose level, and 150 and 50 mg/kg bw/day were selected for middle and low dose levels, respectively, using a common ratio of about 3.
- Rationale for animal assignment: Successfully copulated females were assigned to the test groups on the day of copulation using the system package software for safety study (LATOX-F/V5, FFC), on the basis of the body weight on Day 0 of gestation. Animals copulated on the same day were uniformly assigned to the groups.
- Fasting period before blood sampling for (rat) dam thyroid hormones: Not reported
- Time of day for (rat) dam blood sampling: Not reported

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (before and after each administration) during the administration period and once daily during the rest of the experimental period.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on Days 0, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 of gestation.
Body weight gain during Day 0 to 5, 5 to 20 and 0 to 20 of gestation were calculated.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined : Yes
- Time schedule: The amount of food including the feeder was measured on Days 0, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 of gestation. However, only the supplied food was measured on Day 0 of gestation, and only the remaining food on Day 20 of gestation. The mean daily food consumption was calculated using the weights of supplied and remained diets.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Number of dead fetuses: Yes
- Other: Regarding the number of resorbed and dead fetuses, the following was specified:
*the number of early dead embryos (deciduoma),
*number of late dead embryos (morphologically indistinct dead embryos with placenta and amnion)
* number of dead fetuses (including macerated fetuses) were recorded.

On the basis of the body weight on the day of necropsy, their relative weights were calculated.
Relative weight: (Absolute weight/body weight on the day of necropsy) × 100

Based on the implantation rate, live fetus rate and resorbed and dead fetus rate were calculated.

The placentas of live fetuses were weighed after gross examination for anomalies. The mean placental weight for each sex per litter was calculated. Placentas were discarded after examination.

The uterus which did not have conceptuses was stained with 10 vol% ammonium sulfide solution to confirm the implantation sites.

Two animals without implantation were designated as unsuccessfully copulated (infertile) animals. After the intrauterine examinations, ovaries and uterus were discarded.

Calculations:
Implantation rate:
(Number of implantations/number of corpora lutea graviditatis) × 100

Live fetus rate:
(Number of live fetuses/number of implantations) × 100

Resorbed and dead fetus rate:
(Number of dead embryos and fetuses/number of implantations) × 100

Blood sampling:

- Plasma: No
- Serum: Yes
- Volume collected: Not reported
- Blood samples were collected from the abdominal aorta of dams under isoflurane anesthesia. Blood was processed to serum and stored deep-frozen until analysis.
- The following parameters were determined in serum by ELISA: Thyroxine (T4), Triiodothyronine (T3) and Thyroid-stimulating hormone (TSH).
- Other: The all data of one animal were excluded from the hormone calculation, because this animal had an abortion.

Fetal examinations:

- External examinations: Yes: [all per litter]
Incidence of external abnormalities: (Number of abnormal fetuses / number of fetuses observed) × 100
- Soft tissue examinations: Yes: [half per litter]
Incidence of visceral abnormalities: (Number of abnormal fetuses / number of fetuses observed) × 100
- Skeletal examinations: Yes: [half per litter]
Ad skeletal examinations: Abnormalities were classified as skeletal malformations and variations, and the degree of ossification (number of ossified bones in the cervical centrum, number of incompletely ossified and unossified sternebra, number of ossified bones in metacarpal, fore proximal phalanx, middle phalanx, distal phalanx, metatarsal, hind proximal phalanx, middle phalanx and distal phalanx, number of ossified bones in sacrococcygeal vertebra and other regions with incomplete ossification or unossification). The incidences of skeletal malformation and skeletal variation were calculated. The degree of ossification (countable case) was evaluated as representing values of ossification and the others as the incidence of incomplete ossification.
Incidence of skeletal malformation: (Number of fetuses with malformation / number of fetuses observed) × 100
Incidence of skeletal variation: (Number of fetuses with variation / number of fetuses observed) × 100
Incidence of incomplete ossification: (Number of abnormal fetuses/ number of fetuses observed) × 100
- Head examinations: Head and abdomen were microscopically examined according to the modified Wilson method and their thorax was examined according to Nishimura’s microscopic necropsy method.
- Anogenital distance of all live rodent pups: Yes
- Sex determination and body weight: Sex ratio was determined; Each fetus was weighed individually
Sex ratio: (Surviving male fetuses / surviving male and female fetuses) × 100

Statistics:

The body weight, body weight gain, food consumption, organ weights (absolute and relative), hormones, number of corpora lutea graviditatis, number of implantations, number of live fetuses, number of resorbed and dead fetuses (number of implantation sites), AGD, fetal weight, placental weight and degree of ossification were analyzed by Bartlett’s test for equality of variance. When the Bartlett’s test showed homoscedasticity (not significantly different), the data were analyzed by Dunnett’s multiple comparison test to assess the statistical significance of difference between the control group and each test substance-treated group. When the Bartlett’s test showed heteroscedasticity (significantly different), the data were analyzed by the Steel’s test to assess the statistical significance of difference between the control group and each test substance-treated group.
The implantation rate, live fetus rate, resorbed and dead fetus rate, sex ratio, and incidences of external anomalies, visceral abnormalities, skeletal variations, skeletal malformations and incomplete ossification were analyzed by Steel’s test to assess the statistical significance of difference between the control group and each test substance-treated group.
The significance level of the Bartlett's test was two-sided 5%. The significance levels of the other tests were two-sided 5% and 1%. No statistical tests were used for clinical signs and gross and histopathological findings.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 500 mg/kg bw/day group, it was found that one dam had an abortion at intrauterine examination on Day 20 of gestation. This aborted animal showed irregular respiration, abnormal respiratory noise, smudge of perinasal area and loose stool. Furthermore, the animal showed little or no food consumption on several days from gestation day 9 onwards and body weight gain was significantly decreased. During macroscopic evaluation an atrophic thymus and black patches in the stomach were observed. Based on these findings the abortion was likely a secondary effect of the poor general condition of the animal's due to treatment effects. The findings for this animal were not included in total of group because the definite day of abortion was unknown.
In other dams in the 500 mg/kg bw/day group, loose stool was observed in 19/20 animals.
In addition, loose stool (dark green) and mucous feces (dark green) were observed in one animal. In the 150 mg/kg bw/day group, loose stool was observed in 3/23 animals. In the 50 and 500 mg/kg bw/day groups, irregular respiration was observed in 1/23 and 2/20 animals, respectively. However, this finding was considered to be incidental because it was found in a few animals.

Mortality:

no mortality observed

Description (incidence):

No animals died in any group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 500 mg/kg bw/day group, the mean body weights from Day 7 to 20 of gestation, body weight gains from Day 5 to 20 and from Day 0 to 20 of gestation were significantly lower than those in the control group. Moreover, the mean body weights from Day 6 to 10 of gestation were decreased from Day 5 of gestation (start day of administration). In the 50 and 150 mg/kg bw/day groups, there were no statistically significant differences in the body weights or body weight gains when compared with control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 500 mg/kg bw/day group, the mean daily food consumption was significantly lower than that in the control group from Day 6 to 11 and from Day 16 to 20 of gestation. In the 150 mg/kg bw/day group, significantly lower daily food consumption was observed on Day 6 of gestation. However, this change was considered to be not related to the test substance treatment because it was slight change and no changes were observed in other measurement days.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

The T4 level in the 150 mg/kg bw/day group was significantly lower than that in the control (0.8x control).
There were no statistically significant differences in the levels of T3 and TSH between each treatment group and control group.
The effect on T4 level was not considered to be toxicologically relevant due to the limited effect size and absence of concomitant statistically significant changes in the TSH level and histopathological changes in the thyroid gland.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the absolute and relative weights of thyroid glands and gravid uterus between treatment groups and control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the macroscopic examination of females on Day 20 of gestation, thickening of forestomach was observed in 9/23 and 20/20 dams, and red patches on forestomach were observed in 2/23 and 3/20 dams, in the 150 and 500 mg/kg bw/day groups, respectively. These findings were also noted in the dose range finding study given 500 mg/kg/day or more. Therefore, these findings and changes were considered to be test substance-treatment related effects.
Moreover, in the 500 mg/kg bw/day group, atrophic thymus, black patches in the stomach or dilated lumen of the small intestine was observed in one dam each. However, these findings were not considered to be a direct effect of test substance-treatment because these were observed in small number of animals including an aborted animal.
In the aborted animal of 500 mg/kg bw/day group, atrophic thymus, thickening of forestomach, black patches and red patches in the stomach were observed. From these findings, it was considered that this animal showed debility condition. Therefore, abortion was judged a secondly effect related the deterioration of general condition due to treatment effects, such as decreased food consumption and reduced body weight gain.
Also in the non-pregnant animals, thickening of forestomach and red patches on forestomach were observed.
In one dam of the 150 mg/kg bw/day group, absence of the kidney (left), enlarged kidney (right) and defect of uterus were observed. However, these absent or defective findings were considered to be congenital anomalies, and enlarged kidney was considered compensated effect against absent of one side. All the other findings, brownish lungs, brown patches in the lungs, nodule in the stomach and focal depression in the kidneys were observed sporadically in all groups. These findings were often observed in the rats of this strain, therefore, these were judged to be spontaneous occurring lesions.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No abnormal findings were noted in the thyroid glands in any groups.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No abnormal findings were noted in the thyroid glands in any groups.

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

In the uterine examination on Day 20 of gestation, one animal in the 500 mg/kg/day group was judged to be aborted during the gestation period. Number of corpora lutea and implantations for this animal were included in calculations however, other data of this animal, such as findings of clinical signs and gross findings, values of body weight, food consumption and hormones were excluded. In addition, organ weights measurement and histopathological examination of thyroid glands were not conducted.
This animal showed a little or no food consumption on several days later from Day 10 of gestation, and body weight gain during the administration period was significantly decreased. In addition, irregular respiration, abnormal respiratory noise, smudge of perinasal area and loose stool were observed as clinical findings. In macroscopic examination of this animal, atrophic thymus and black patches in the stomach were observed. Based on these findings the abortion was likely a secondary effect of the poor general condition of the animal's due to treatment effects.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

In the 500 mg/kg bw/day group, the mean placental weights of male and female fetuses were significantly lower than those in the control group. This change was considered to be secondary effects caused by suppression of body weight gain and lower food consumption of dams in this group.

In the observation of placentas, enlarged placenta was observed in 2 and 1 dam in the control and 50 mg/kg bw/day groups, respectively. However, the enlarged placenta was not considered to be test substance treatment-related effect because there were no abnormal findings in the 150 and 500 mg/kg bw/day groups.

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the 500 mg/kg bw/day group, the mean female fetus weight was significantly lower than that in the control group (0.93x of control). The mean male fetus weight in this group tended to be lower compared to the control group (0.96x of control). However, these changes were considered to be likely secondary effects related to suppression of body weight gain and lower food consumption of dams.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the sex ratio.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There we not statistically significant differences in the AGD between each treatment group and control group.

External malformations:

no effects observed

Description (incidence and severity):

External abnormalities were noted in 0 (0.0%), 0 (0.0%), 1 (0.4%) and 0 (0.0%) fetuses for the control, 50, 150 and 500 mg/kg bw/day groups, respectively. There were no statistically significant differences in the incidences of fetuses with external abnormalities between each treatment group and control group. As the finding, gastroschisis was observed in one fetus of 150 mg/kg bw/day group. However, this finding was not considered to be test substance treatment-related, because this finding was observed in only one fetus and not observed in the 500 mg/kg bw/day group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted in 3 (1.8%), 1 (0.6%), 3 (2.0%) and 5 (3.2%) fetuses in the control, 50, 150 and 500 mg/kg bw/day groups, respectively. There were no statistically significant differences in the incidences of fetuses with skeletal malformations between each treatment group and control group. Findings included absent rib, fused rib, short rib, misshapen sternebra, absent thoracic vertebra and absent lumbar vertebra in the control or treatment groups, but there were no statistical significance in the incidences of these findings.
Skeletal variations were noted in 11 (9.6%), 15 (8.4%), 7 (4.3%) and 6 (3.7%) fetuses in the control, 50, 150 and 500 mg/kg/day groups, respectively. There were no statistically significant differences in the incidences of fetuses with skeletal variations between each treatment group and control group. Findings included cervical rib, discontinuous rib, short supernumerary rib and sacralization of lumbar vertebra in the control or treatment groups, but there were no statistical significances in the incidences of these findings.
Retarded ossification was noted in 108 (62.2%), 93 (58.9%), 97 (55.9%) and 92 (62.4%) fetuses in the control, 50, 150 and 500 mg/kg bw/day groups, respectively. There were no statistically significant differences in the number of fetuses with retarded ossification or in the incidences of findings between each treatment group and control group.
Findings included craniofenestria, incomplete ossification of the frontal, incomplete ossification of the hyoid, unossified hyoid, incomplete ossification of the interparietal, incomplete ossification of the nasal, incomplete ossification of the parietal, incomplete ossification of the squamosal, bipartite ossification of the supraoccipital, incomplete ossification of the supraoccipital, incomplete ossification of the zygomatic, bipartite ossification of the sternebra, incomplete ossification of the cervical arch, bipartite ossification of the thoracic centrum, dumbbell ossification of the thoracic centrum, incomplete ossification of the thoracic centrum, unossified thoracic centrum, dumbbell ossification of the lumbar centrum, incomplete ossification of the lumbar centrum, incomplete ossification of the sacral arch, unossified sacral arch, unossified sacral centrum, incomplete ossification of the caudal centrum, incomplete ossification of the ischium, incomplete ossification of pubis and unossified pubis.
In the 500 mg/kg bw/day group, the numbers of unossified sternebra and incomplete ossification of sternebra were significantly higher, and the number of sacrococcygeal vertebra was significantly lower, than those in the control group. There were no statistically significant differences in numbers of ossified metacarpal, metatarsal, fore/hind proximal, middle and distal phalanx, and cervical vertebral centrum between each treatment group and control group. The findings in the skeletal examinations were not considered to be treatment-related, because the incidences of changes were low or similar incidences were noted in the control group.

Visceral malformations:

no effects observed

Description (incidence and severity):

Visceral abnormalities were noted in 38 (27.8%), 25 (15.4%), 31 (20.0%) and 31 (22.5%) fetuses in the control, 50, 150 and 500 mg/kg bw/day groups, respectively. There were no statistically significant differences in the incidences of fetuses with visceral abnormalities between each treatment group and control group. Findings included thymic remnant in the neck, persistent left umbilical artery, abnormal origin of the left common carotid artery, small nasal cavity, misshapen lung, abnormal liver lobation, misshapen liver, small spleen, dilated renal pelvis, convoluted ureter, dilated ureter, anorchia, malpositioned testis, absent epididymis, misaligned palate rugae, misshapen palate rugae and bilateral azygos veins in the control or treatment groups, but there was no statistical significance in the incidences of these findings. The findings in the visceral examinations were not considered to be treatment-related, because the incidences of changes were low or similar incidences were noted in the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of toxicological relevant effects at dose levels up to and including the highest dose levels tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

ANALYSIS OF DOSING FOMULATION

The results of stability/homogeneity analyses confirmed the stability of the prepared dosing formulations of 10 and 200 mg/mL, since the remaining values of the samples after storage were within the acceptance limit (within 100.0±10.0%). The RSDs were less than 5.0% in all the dosing formulations. These results demonstrated that the test substance in corn oil was stable and homogeneous after 4 days of storage in a tight light-resistant container under refrigeration and subsequent 6 hours of storage in a tight light-resistant container at room temperature. On the other hand, the analytical results for stored 8 days were not satisfied the criterion (within 100.0±10.0%) in 10 mg/mL formulation.
The results of the concentration analyses of NBDI formulations confirmed the preciseness of the prepared dosing formulations of 12.5, 37.5 and 125 mg/mL, since each analytical value of the samples was within the acceptance limit (within 100.0±10.0% of nominal concentration). The RSDs were less than 5.0% in all the dosing formulations. No interference peak was detected at the retention time of NBDI on the chromatogram of the dosing formulation for the control samples. These results satisfied the criteria for the concentration/homogeneity of the dosing formulations.

 

SUMMARY TABLE RESULTS

Dose level (mg/kg bw/day)	0	50	150	500
Pregnant/total dams	23/23	23/23	23/23	21/23
Dams with live fetuses	23	23	23	20
Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses	0	0	0	1
Mean number of corpora lutea	16.3	16.1	16.2	16.0
Mean number of implantations	15.4	15.2	15.3	15.2
-early resorptions -late resorptions (total and percent)	21 (7.8%) 0 (0.0%)	25 (6.8%) 0 (0.0%)	17 (4.9%) 3 (0.8%)	23 (7.7%) 3 (1.0%)
Body weight on day 20 (g)	424.3	423.3	418.4	381.6**
Body weight gain day 5-20 (g)	130.2	126.8	123.5	86.2**
Gravid uterine weight (g and percent)	78.34 (18.34%)	75.94 (17.73%)	77.98 (18.58%)	69.93 (18.24%)
Mean live offspring (number)	14.5	14.1	14.4	14.0
Live offspring (total and percent)	333 (92.2%)	324 (92.9%)	332 (94.3%)	280 (91.3%)
Sex ratio (male%)	54.3	50.3	54.2	50.1
Anogenital distance males (mm)	2.9	2.8	2.9	2.8
Anogenital distance females (mm)	1.3	1.2	1.3	1.3
Mean fetal body weight males (g)	3.72	3.77	3.80	3.56
Mean fetal body weight females (g)	3.60	3.50	3.56	3.36*
Placental weight males (g)	0.46	0.47	0.45	0.40**
Placental weight females (g)	0.46	0.47	0.43	0.38*
Skeletal malformations number and percent of fetuses	3 (1.8%)	1 (0.6%)	3 (2.0%)	5 (3.2%)
Variations (total and percent) -external -soft tissue -skeletal	0 38 (27.8%) 11 (9.6%	0 25 (15.4%) 15 (8.4%)	1(0.4%) 31 (20.0%) 7 (4.3%)	0 31 (22.5%) 6 (3.7%)

*: significantly different from the control group at 0.05

**: significantly different from the control group at 0.01

Conclusions:

A developmental toxicity study was performed according to OECD TG 414 and in accordance with GLP principles. The NOAEL of NBDI for maternal toxicity was judged to be 150 mg/kg bw/day, based on the thickening of the forestomach and red patches on the forestomach in combination with reduced body weight (gain) and food consumption in the 500 mg/kg bw/day group. The NOAEL for reproductive function in the dams was judged to be >= 500 mg/kg bw/day, because there were no clear effects on the reproductive parameters. The NOAEL for embryo-fetal development was judged to be >= 500 mg/kg bw/day, because there were no toxicological relevant effects on any of the developmental parameters.

Executive summary:

A developmental toxicity study was executed according to OECD TG 414 and in accordance with GLP principles. This study was performed to assess the potential maternal toxicity and embryo fetal toxicity of NBDI. The dosing solutions (vehicle: corn oil) of NBDI were administered to the groups of mated female Crl:CD(SD) rats, consisting of 23 animals per group, by oral gavage at dose levels of 0, 50, 150 or 500 mg/kg bw/day from Day 5 to 19 of gestation. The clinical signs, body weight and food consumption of the dams were recorded regularly during the gestation period. On Day 20 of gestation, the dams were sacrificed and necropsied (cesarean section), and the thyroid glands and gravid uterus weights were measured. The thyroid glands of dams were histopathologically examined. The serum levels of thyroxine (T4), triiodothyronine (T3) and thyroid-stimulating hormone (TSH) were measured. The fetuses were examined for their viability, growth and morphological changes. The results are summarized as follows.

Effects on the dams

No dead animals were found during the gestation period.
One aborted animal was observed in the 500 mg/kg bw/day group. This aborted animal showed decreased food consumption and reduced body weight gain. During the macroscopic evaluation an atrophic thymus and black patches in the stomach were observed. It is likely that the abortion is a secondary effect.
Test substance treatment-related effects were noted in the 150 and 500 mg/kg bw/day groups.
As the clinical signs, loose stool was observed in the 150 and 500 mg/kg bw/day groups. In the 500 mg/kg bw/day group, lower food consumption was observed during the administration period. Body weights from Day 6 to 10 of gestation were decreased from Day 5 of gestation (start day of administration), lower body weights and suppressed body weight gains were observed during the administration period in the 500 mg/kg bw/day group. In the macroscopic examination, thickening of forestomach and red patches on forestomach were observed in the 150 and 500 mg/kg bw/day groups.
There were no statistically significant changes in the weights of thyroid glands and gravid uterus in any treatment groups.
The histopathological examination of thyroid glands revealed no abnormal findings in any treatment group.
There were no treatment-related effects in the levels of T4, T3 and TSH in any treatment groups.

Lower placental weights were observed in the 500 mg/kg bw/day group. However, this change was considered to be a likely secondary effect caused by suppression of body weight gain and lower food consumption of dams.
There were no changes in the number of corpora lutea, number of implantations, number of resorptions and dead fetuses, live fetus rate, the resorptions and dead fetus rate or placental observations in any group.
In the 50 mg/kg bw/day group, there were no obvious treatment-related effects on dams.

Effects on fetuses

In the 500 mg/kg bw/day group, the mean fetus weight of females was lower, and males showed lower tendency. However, these changes were considered to be likely secondary effects caused by suppression of body weight gain and lower food consumption of dams. There were no test substance treatment-related effects on the sex ratio, anogenital distance (AGD), findings of external, visceral nor skeletal examinations.

In conclusion, the no-observed-adverse-effect level (NOAEL) of NBDI for maternal toxicity was judged to be 150 mg/kg bw/day, based on the thickening of the forestomach and red patches on the forestomach in combination with reduced body weight (gain) and food consumption in the 500 mg/kg bw/day group. The NOAEL for reproductive function in the dams was judged to be >= 500 mg/kg bw/day, because there were no clear effects on the reproductive parameters. The NOAEL for embryo-fetal development was judged to be >= 500 mg/kg bw/day, because there were no toxicological relevant effects on any of the developmental parameters.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was performed according to GLP and OECD test guideline (Klimisch score 1).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A developmental toxicity study was executed according to OECD TG 414 and in accordance with GLP principles. This study was performed to assess the potential maternal toxicity and embryo fetal toxicity of NBDI. The dosing solutions (vehicle: corn oil) of NBDI were administered to the groups of mated female Crl:CD(SD) rats, consisting of 23 animals per group, by oral gavage at dose levels of 0, 50, 150 or 500 mg/kg bw/day from Day 5 to 19 of gestation. The clinical signs, body weight and food consumption of the dams were recorded regularly during the gestation period. On Day 20 of gestation, the dams were sacrificed and necropsied (cesarean section), and the thyroid glands and gravid uterus weights were measured. The thyroid glands of dams were histopathologically examined. The serum levels of thyroxine (T4), triiodothyronine (T3) and thyroid-stimulating hormone (TSH) were measured. The fetuses were examined for their viability, growth and morphological changes. The results are summarized as follows.

Effects on the dams

No dead animals were found during the gestation period.
One aborted animal was observed in the 500 mg/kg bw/day group. This aborted animal showed decreased food consumption and reduced body weight gain. During the macroscopic evaluation an atrophic thymus and black patches in the stomach were observed. It is likely that the abortion is a secondary effect.
Test substance treatment-related effects were noted in the 150 and 500 mg/kg bw/day groups.
As the clinical signs, loose stool was observed in the 150 and 500 mg/kg bw/day groups. In the 500 mg/kg bw/day group, lower food consumption was observed during the administration period. Body weights from Day 6 to 10 of gestation were decreased from Day 5 of gestation (start day of administration), lower body weights and suppressed body weight gains were observed during the administration period in the 500 mg/kg bw/day group. In the macroscopic examination, thickening of forestomach and red patches on forestomach were observed in the 150 and 500 mg/kg bw/day groups.
There were no statistically significant changes in the weights of thyroid glands and gravid uterus in any treatment groups.
The histopathological examination of thyroid glands revealed no abnormal findings in any treatment group.
There were no treatment-related effects in the levels of T4, T3 and TSH in any treatment groups.

Lower placental weights were observed in the 500 mg/kg bw/day group. However, this change was considered to be a likely secondary effect caused by suppression of body weight gain and lower food consumption of dams.
There were no changes in the number of corpora lutea, number of implantations, number of resorptions and dead fetuses, live fetus rate, the resorptions and dead fetus rate or placental observations in any group.
In the 50 mg/kg bw/day group, there were no obvious treatment-related effects on dams.

Effects on fetuses

In the 500 mg/kg bw/day group, the mean fetus weight of females was lower, and males showed lower tendency. However, these changes were considered to be likely secondary effects caused by suppression of body weight gain and lower food consumption of dams. There were no test substance treatment-related effects on the sex ratio, anogenital distance (AGD), findings of external, visceral nor skeletal examinations.

In conclusion, the no-observed-adverse-effect level (NOAEL) of NBDI for maternal toxicity was judged to be 150 mg/kg bw/day, based on the thickening of the forestomach and red patches on the forestomach in combination with reduced body weight (gain) and food consumption in the 500 mg/kg bw/day group. The NOAEL for reproductive function in the dams was judged to be >= 500 mg/kg bw/day, because there were no clear effects on the reproductive parameters. The NOAEL for embryo-fetal development was judged to be >= 500 mg/kg bw/day, because there were no toxicological relevant effects on any of the developmental parameters.

Justification for classification or non-classification

Based on the currently available data, NBDI is not classified for adverse effects on reproduction and development according to the CLP Regulation (EC) No. 1272/2008.