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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May - October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
NA

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other:
Version / remarks:
Test Method Concerning New Chemical Substances, No.1221-1, Pharmaceutical and Medical Safety Bureau, MHLW, No. 1, Manufacturing Industries Bureau, METI (December 09, 2015), No. 1512211, Environmental Policy Bureau, MOE, (December 21, 2015)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
white fine powder
Specific details on test material used for the study:
Identity: 4,4'-Oxybis (benzenesulfonyl hydrazide) (OBSH)
CAS No.: 80-51-3
EC No.: 201-286-1
Code No: K-5450
Batch/Lot No:.201512001
Appearance: White fine powder
Purity 99.5~100%
Storage conditions: Room temperature, Protected from light, Protected from moisture
Expiry date: December, 2016

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Species Rat (Specific Pathogen Free)
Strain/Substrain Sprague-Dawley/Crl:CD (SD)
Number at receipt Female 120
Number on study Female 88

Age at receipt Approximately 9 weeks
Age on dosing start Approximately 11 weeks
Body weight range 254.1-303.5 g at the initiation of dosing

Supplier Orient Bio Inc. 322, Galmachi-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do 13201, Republic of Korea
Method of identification Each animal was individually identified using an oil marker during acclimation period, and tail tattoo method after group assignment.
Acclimation period An acclimation period of 7 days was permitted between receipt of the animals and the start of mating to accustom the rats to the animal room environment. During this acclimation period, all animals were observed once a day for any Clinical signs of disease. Healthy (specific pathogen free) animals were used in this study and the data for the health monitoring from the animal supplier was included in the raw data.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The test item was suspended in the vehicle to reach the target concentration of the highest dose. The lower doses of the test item were prepared by serial dilution of the higher dose formulation with the vehicle. The formulations were dispensed into light-protective tubes for use and then stirred on a magnetic stirrer at room temperature until the completion of formulation.

DIET PREPARATION
- Rate of preparation of diet (frequency):
Dose formulations were prepared once per week and stored at room temperature

- Mixing appropriate amounts with (Type of food):
The dose volume (2 mL/kg) for each animal was calculated using the most recent body weight measurement. Oral administration of the test item was performed by gavage. On the day of dosing, the formulated test item was stirred for at least 10 minutes by magnetic stirrer prior to use and constantly stirred throughout the dosing in the animal room.

- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil shown to be a suitable vehicle for OBSH formulations
- Concentration in vehicle: 0, 10, 30 and 80 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg
- Lot/batch no. (if required): MKBS6944V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analysis including concentration and homogeneity tests was performed before the first formulations and additional concentration analysis was conducted for the last formulations.
The concentrations of all the dosing formulations were 92.2 % to 103.4 % within ± 10 % of the nominal concentration and CV (Coefficient of Variation) were 0.1 to 1.7 within 5 %. The results were all in acceptable range.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male: 2 or 1 female
- Length of cohabitation:
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
Gestation day (GD) 6 to 20
Frequency of treatment:
Daily
Duration of test:
21 days. The pregnant female rats were sacrifized at caesarean section on gestation day 21.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1: Control group
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
G2
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
G3
Dose / conc.:
160 mg/kg bw/day (nominal)
Remarks:
G4
No. of animals per sex per dose:
22 pregnant females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
To determine dose levels of this definitive study, a Dose Range-Finding Prenatal Developmental Toxicity Study (KIT Study No. N116010) was conducted at dose levels of 0, 10, 30 and 100 mg/kg/day. No treatment-related changes were observed in any examined parameters including mortality, clinical observation, body weight/weight change, food consumption, macroscopic lesions, caesarean section data, and fetal weight.

Data available from a 28D repeated dose toxicity study in rats conducted at dose levels of 0, 10, 30, 100 and 200 mg/kg/day, all animals at the highest dose level died or were sacrificed due to moribund until dosing days 27 in females. At 100 mg/kg/day in females, food consumption was decreased, and weights of liver and kidney were increased. At histopathological examination, fatty degeneration of proximal tubular epithelium was noted in the kidney at 100 mg/kg/day.
Additionally, in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test which was conducted at dose levels of 0, 5, 15 and 45 mg/kg/day1), in female rats, weights of liver and kidney were increased at 45 mg/kg/day, and no reproductive and developmental toxicities were observed at any doses tested.

Based on these results, doses of 0 (vehicle), 20, 60 and 160 mg/kg/day was selected for the main OECD 414 study. The 20 mg/kg/day dose expected to be a no-observable-adverse-effect-level (NOAEL) for both maternal and embryo-fetal toxicity, and the 160 mg/kg/day dose is expected to produce maternal toxicity

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes:
- Time schedule: Clinical observations were recorded three times a day on the days of treatment and once a day on non-treatment days

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GD 0, 3, 6, 9, 12, 15, 18, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption were recorded on GD 0, 3, 6, 9, 12, 15, 18, and 21.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined:
A gross observation of the cranial, thoracic, abdominal and pelvic viscera was performed. Further the dams were examined for the number and distribution of corpora lutea, implantation sites and uterine contents. Fetuses and placentas were weighed and examined for gross external alterations.

OTHER:
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 early resorptions: Yes
- Number of late resorptions: Yes
- Other: live/dead fetuses
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [even number fetuses ]
- Skeletal examinations: Yes: [odd number fetuses ]
- Head examinations: Yes: [even number fetuses]

Live fetuses retrieved from each gravid uterus were examined immediately to evaluate external abnormalities. Fetuses were numbered from the left uterine horn to the right. Alternate fetuses (odd numbers: skeletal examination, even numbers: visceral examination) were selected for either skeletal or visceral examination.
For visceral examination, live fetuses were fixed with Bouin’s solution, and then modified Wilson's technique was used for the head, Nishimura method for the thorax and modified Staples method for the abdomen. After examination, visceral specimens were identified and preserved per litter.
For skeletal examination, after removing skin, fetuses were fixed with 95% ethyl alcohol. The evaluation of skeletal abnormalities was performed after Alcian blue and Alizarin Red S staining. After examination, skeletal specimens were identified and kept per litter.
Statistics:
Statistical analyses were performed by comparing the vehicle control group with the treatment groups using Pristima and Statistical Analysis Systems.
Data from the treated groups was compared statistically with those of the control group using the following methods. The data was analyzed for homogeneity of variance using Bartlett's test. Homogeneous data was analyzed using the Analysis of Variance and the significance of inter-group differences between the control and treated groups was analyzed using Dunnett's t test. Heterogeneous data was analyzed using Kruskal-Wallis test and the significance of inter-group differences between the control and treated groups was assessed using Dunn's Rank Sum test.
One-way analysis of covariance (ANCOVA) was used to analyze fetal and placental weight data. The litter size was used as the covariate. Litter data were statistically evaluated using the statistical unit as a litter.
The fetal and litter incidence of findings was analyzed using χ2-test followed by the Fisher's exact test where necessary.
Numerical data obtained during the conduct of the study was subjected to calculation of group means and standard deviations and reported in the final report. Data was considered to be significant when p<0.05 or p<0.01.
Historical control data:
yes: See attached document below with KIT historical control data
Test Facility: Korea Institute of Toxicology (KIT)
Period: 2013. 01 – 2016. 05 (Only definitive studies are included in the KIT Historical Control Data)
Animal Strain Crl: CD(SD) rats
Supplier:Orient Bio. Ltd., Republic of Korea

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related clinical signs were observed in the 160 mg/kg/day group (See attached document below with summary tables - Table 2). Soiled perineal region, irregular respiration, subdued behavior and thin appearance were observed in 4, 4, 2 and 3 dams, respectively, in late gestation period (GD 18 - GD 21), and these clinical signs were considered adverse since the incidence was high (Animal ID 68, 69, 72, 77).

Salivation was observed in the 60 and 160 mg/kg/day groups, and it was treatment-related since the incidence was high. However, it was not considered adverse since it was found only immediately after dosing and then disappeared, and it may be attributed to the irritating effect of the test item since no adverse effect was found in salivary gland in previous repeated dose toxicity studies.

Other clinical sign, loss of fur, observed in the test item-treated groups was considered incidental since the incidence was comparable to that of controls.

(See attached draft report, Appendix 2).
Description (incidence and severity):
NA
Mortality:
mortality observed, treatment-related
Description (incidence):
Treatment-related death occurred in one animal (Animal No.: 68) on GD 21 (necropsy day) in the 160 mg/kg/day group (See attached document below with summary tables - Table 1) . The dead animal showed soiled perineal region, irregular respiration, subdued behavior and thin appearance between GD 18 and GD 20. Body weight of this animal was continuously increased from 261.0 g on GD 6 (dosing start day) to 283.9 g on GD 12, but was dramatically reduced to 227.6 g on GD 18. In addition, a marked decrease in daily food consumption was observed at intervals of GD 12-15 (14.3 g) and GD 15-18 (3.7 g), compared to other animals of the same dosing group (Summary tables 3-5). However, at necropsy, no gross lesion was found in this animal. In this study, the cause of death was not determined, but animal death was considered dose related since severe maternal toxicity was observed and, in a previous repeated dose toxicity study (28D), animal death or a moribund state occurred in females at 200 mg/kg/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A treatment-related decrease in mean body weight and weight gain was observed in the 160 mg/kg/day group during treatment period (GD 6 - GD 21), and mean body weight and weight gain were decreased to the ranges of 88.1 % - 97.5 % and 55.3% - 62.0 % of controls, respectively. These decreases were considered to be adverse and be resulted from reduction of food intake during treatment period. No treatment-related change in body weight was observed in the 20 and 60 mg/kg/day groups (See attached document below with summary tables - Table 3-5).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A treatment-related decrease in food consumption was observed in the 160 mg/kg/day group during the treatment period, and its range was 75.2 % - 86.7 % of controls. This decrease was considered to be an adverse effect of the test item on maternal food consumption with decreased mean body weight and weight gain at this period. No treatment-related change in food consumption was observed in the 20 and 60 mg/kg/day groups (See attached document below with summary tables - Table 5).
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A treatment-related decrease in gravid uterine weight, corrected terminal body weight and net body weight change was observed in the 160 mg/kg/day group with 82.2 %, 90.1 % and 19.9 % of controls, respectively. A decrease in mean body weight on GD 21 was resulted from a decrease in maternal weight gain as well as gravid uterine weight including decreased fetal weight (See attached document below with summary tables - Table 7).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related macroscopic lesions were found in kidneys and adrenal glands in the 160 mg/kg/day group. Pale discoloration in kidney and enlarged adrenal gland were observed in 3 and 2 dams, respectively. However, it could not be determined in this study if these findings were adverse or not, since histopathological observation and/or measurement of serum biochemistry parameters were not conducted and in previous repeated toxicity studies these findings were not observed. Other macroscopic findings were considered incidental sine the incidence was low and/or there was no dose-relation ( (See attached document below with summary tables - Table 6).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Non-pregnancy occurred in 1 female in the 0 and the 60 mg/kg/day group, and the number of pregnant dams was 21, 22, 21 and 22 in the 0, 20, 60 and 160 mg/kg/day groups, respectively (See Table 1 in attached document below).

Treatment-related death occurred in one animal (Animal No.: 68) on GD 21 (necropsy day) in the 160 mg/kg/day group. The dead animal showed soiled perineal region, irregular respiration, subdued behavior and thin appearance between GD 18 and GD 20. Body weight of this animal was continuously increased from 261.0 g on GD 6 (dosing start day) to 283.9 g on GD 12, but was dramatically reduced to 227.6 g on GD 18. In addition, a marked decrease in daily food consumption was observed at intervals of GD 12-15 (14.3 g) and GD 15-18 (3.7 g), compared to other animals of the same dosing group. However, at necropsy, no gross lesion was found in this animal. In this study, the cause of death was not determined, but animal death was considered dose related since severe maternal toxicity was observed and, in a previous repeated dose toxicity study (28D oral), animal death or a moribund state occurred in females at 200 mg/kg/day (See IUCLID section 7.5.1).


.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortions as well as early deliverables and still births were seen. Total litter loss was observed in two dams of the 160 mg/kg/day group, one dam found dead and the other dam showing severe maternal toxicity.Total litter loss was observed in two dams of the 160 mg/kg/day group, one dam found dead and the other dam showing severe maternal toxicity.
(See attached document below with summary tables and also draft report).
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No statistical signifficant effect were seen. However:
a 2.3 fold post-implantation loss was seen at 160 mg/kg bw/day when compared to control group, i.e means were 13.2% and 5.7%, respectively.
a 2.1 fold pre-implantation loss was seen at 160 mg/kg bw/day when compared to control group, i.e means were 6.6% and 3.2%, respectively.
See attached document below with summary tables (Table 8).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The total litter loss occured mainly due to dead fetuses, not resorption, indicating that fetuses died in late gestation period:

Total litter loss was observed in two dams of the 160 mg/kg/day group ( animal ID 68 which was found dead and animal ID 69). It was found in dams showing severe maternal toxicity (See attached document below with summary tables - Tables 10-15 and the attached draft repprt).

Early or late resorptions:
no effects observed
Description (incidence and severity):
See attached document below with summary tables (Table 8).
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
An increase in the number of dead fetuses was observed in the 160 mg/kg/day group (mean 0.8±3.06) and it was 8-fold and 26.6-fold higher than controls (mean 0.1±0.30) and KIT historical controls (0.03±0.08, max.value: 0.1), respectively (See attached documents below on summary tables - Table 8 and KIT historical control data - Page 281).

The increase in the number of dead fetuses was considered adverse. However, it was not thought to be an direct effect of the test item on fetuses but to be caused by secondary effect since dead fetuses were found only in live dams (animal No. 69, 72, 77) showing severe maternal toxicities in the late gestation period (fetal phase) including abnormal clinical signs such as subdued behavior and thin appearance and reduction of food intake with a marked decrease in body weights, and no treatment-related change was observed in skeletal and visceral examinations except skeletal variations related to retarded ossifications which are indicative to decreased fetal weights (See attached draft report).

An increase in dead fetuses resulted in an increase (2.3-fold over controls) in post-implantation loss and a decrease (89.2% of controls) in live fetuses (litter size) (See attached document below with summary tables).
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
See attached draft report
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): See attached draft report
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Non-pregnancy occurred in 1 female in the 0 and the 60 mg/kg/day group, and the number of pregnant dams was 21, 22, 21 and 22 in the 0, 20, 60 and 160 mg/kg/day groups, respectively (See Table 1 in attached document below).
Details on maternal toxic effects:
Death occurred in one animal (Animal No.: 68) on GD 21 (necropsy day) at 160 mg/kg/day. It was considered dose related since abnormal clinical signs such as soiled perineal region, irregular respiration, subdued behavior, and thin appearance were observed during the late gestation period, and there was a reduction of food consumption including decreased body weight and weight gain in this animal.

In clinical observation, soiled perineal region, irregular respiration, subdued behavior and thin appearance were observed in 4, 4, 2 and 3 dams (animal no 68, 69, 72, 77), respectively, at 160 mg/kg/day in late gestation period (GD 18- GD 21), and these were considered dose related.

A decrease in food consumption was observed at ranges of 75.2 % - 86.7 % of controls at 160 mg/kg/day during the treatment period, and it resulted in decreased mean body weight (88.1 % - 97.5 % of controls) and decreased weight gain (55.3% - 62.0 % of controls). In addition, gravid uterine weight, corrected terminal body weight and net body weight change were decreased to 82.2 %, 90.1 % and 19.9 % of controls, respectively.

See attached document with summary tables and also attached draft report.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 60 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in fetal weights was observed in both sexes of the 160 mg/kg/day group. Mean fetal weight in the 160 mg/kg/day group was 88.2% in both sexes (total value), 88.3 % in males and 88.9 % in females of the control values (See attached document below with summary tables - Table 9). The decrease in fetal weights of both sexes observed in the 160 mg/kg/day group was not considered to be a direct effect of the test item on fetuses, but to be secondary to maternal toxicities including the decrease in food consumption and subsequent body weight reduction.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): A statistically significant decrease in fetal weights was observed in both sexes of the 160 mg/kg/day group (See attached document below with summary tables - Table 9). Mean fetal weight in the 160 mg/kg/day group was 88.2% in both sexes (total value), 88.3 % in males and 88.9 % in females of the control values (See attached document below with summary tables - Table 9). The decrease in fetal weights of both sexes observed in the 160 mg/kg/day group was not considered to be a direct effect of the test item on fetuses, but to be secondary to maternal toxicities including the decrease in food consumption and subsequent body weight reduction.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
An 8-fold increase in dead fetuses resulted in an increase (2.3-fold over controls) in post-implantation loss and a decrease (89.2% of controls) in live fetuses (See attached document below with summary tables - Table 8).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See attached document below with summary tables - Table 8.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
An increase in dead fetuses resulted in a decrease (89.2% of controls) in live fetuses and hence a decrease in litter size.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
See attached document below with summary tables - Table 8.

At caesarean section, a dose related change was observed in the number of dead fetuses at 160 mg/kg/day. This was considered to be a follow of an increase(2.3-fold over controls) in post-implantation loss and a decrease (89.2% of controls) in live fetuses were results of an 8-fold increase in dead fetuses, and the changes were considered adverse. Mean fetal weight at 160 mg/kg/day group was 88.2% in both sexes (total value), 88.3 % in males and 88.9 % in females of the control values. A decrease in fetal weights of both sexes was not considered to be a direct effect of the test item on fetuses, but to be secondary to maternal toxicities including the decrease in food consumption and subsequent body weight reduction.
External malformations:
no effects observed
Description (incidence and severity):
See attached document below with summary tables - Table 10-16.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The data referenced in the following can be found in the attached document below with summary tables (Tables 14-16) and the KIT historical control data can also be found in attached document below.

Incomplete ossification of interparietal and parietal bones and large fontanelle occurred at high incidence (2.08 %, 2.71 % and 4.58 %, respectively) in fetuses of the 160 mg/kg/day group and these skeletal variations were considered to be adverse since they were not observed in the vehicle control group and are beyond limits of KIT historical control values [0.39 % (max.: 1.47 %), 0.83 % (max.: 2.25 %) and 0.27 % (max.: 1.87 %), respectively]. However, these were not thought to be caused by direct effects of the test item on fetuses but indirectly by ossification retardation related to a marked reduction of fetal weights due to maternal toxicities.

The incidence of short supernumerary rib and bipartite and dumbbell ossification of thoracic centrum in fetuses was increased in the test item-treated groups, but it was considered incidental, since it was not dose-related, was comparable to controls and/or was within KIT historical control ranges (See attached document below on KIT historical control data).

No treatment-related change was observed in the number of ossification centers of sternebra, metacarpals, 1st phalanges in both forelimbs, metatarsals, 1st phalanges in both hindlimbs, cervical vertebra, and sacral and caudal vertebra in any of the treatment groups.
Visceral malformations:
no effects observed
Description (incidence and severity):
See attached document below with summary tables - Table 10-16.
Details on embryotoxic / teratogenic effects:
In skeletal malformations, no abnormal finding was observed in any groups.

Incomplete ossification of interparietal and parietal bones and large fontanelle occurred at high incidences (2.08 %, 2.71 % and 4.58 %, respectively) in fetuses of the 160 mg/kg/day group and these skeletal variations were considered adverse since they were not observed in the vehicle control group and are beyond limits of KIT historical control values [0.39 % (max.: 1.47 %), 0.83 % (max.: 2.25 %) and 0.27 % (max.: 1.87 %), respectively]. However, these were not thought to be caused by direct effects of the test item on fetuses but indirectly by ossification retardation related to a marked reduction of fetal weights due to maternal toxicities.

The incidence of short supernumerary rib and bipartite and dumbbell ossification of thoracic centrum in fetuses was increased in the test item-treated groups, but it was considered incidental, since it was not dose-related, was comparable to controls and/or was within KIT historical control ranges (See attached document below with KIT historical control data).

No treatment-related change was observed in the number of ossification centers of sternebra, metacarpals, 1st phalanges in both forelimbs, metatarsals, 1st phalanges in both hindlimbs, cervical vertebra, and sacral and caudal vertebra in any of the treatment groups.


Overall, no teratogenic effects of OBSH observed.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 60 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
other: retarded ossifications

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
160 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Non-pregnancy occurred in 1 female in the 0 and the 60 mg/kg/day group, and the number of pregnant dams was 21, 22, 21 and 22 in the 0, 20, 60 and 160 mg/kg/day groups, respectively (See attached document below with summary tables - Table 1).

Death occurred in one animal (Animal No.: 68) on GD 21 (necropsy day) at 160 mg/kg/day. It was considered dose related since abnormal clinical signs such as soiled perineal region, irregular respiration, subdued behaviour, and thin appearance were observed during the late gestation period, and there was a reduction of food consumption including decreased body weight and weight gain in this animal.  

In clinical observation, soiled perineal region, irregular respiration, subdued behaviour and thin appearance were observed in 4, 4, 2 and 3 dams, respectively, at 160 mg/kg/day in late gestation period (GD 18- GD 21), and these were considered dose related. There were no premature delivery, still births and abortions (See attached document below with summary tables - Table 2 -5).

Applicant's summary and conclusion

Conclusions:
The maternal and embryo-fetal toxicity, and teratogenic potential of 4,4'-Oxybis (benzenesulfonyl hydrazide) (OBSH) following oral administration was evaluated in time mated rats in accordance to OECD 414. OBSH was administrated from gestation day (GD) 6 to 20 at dose levels of 0, 20, 60 and 160 mg/kg/day.The maternal no-observed-adverse-effect level (NOAEL) was considered to be 60 mg/kg/day based on abnormal clinical signs and a decrease in body weight and food consumption at 160 mg/kg/day. The NOAEL for prenatal development was 60 mg/kg/day based on increased number of dead fetuses, decreased fetal weights and retarded ossifications at 160 mg/kg/day.
Executive summary:

The maternal, embryo-fetal toxicity and teratogenic potential of 4,4'-Oxybis (benzenesulfonyl hydrazide) (OBSH) following oral administration was evaluated in time mated Sprague-Dawley/Crl:CD (SD) rats in accordance to OECD 414. OBSH was administrated from gestation day (GD) 6 to 20 at dose levels of 0, 20, 60 and 160 mg/kg/day.

There were no premature delivery, still births and abortions. Total litter loss was observed in two dams of the 160 mg/kg/day group, one dam found dead and the other dam showing severe maternal toxicity. Non-pregnancy occurred in 1 female in the 0 and the 60 mg/kg/day group, and the number of pregnant dams was 21, 22, 21 and 22 in the 0, 20, 60 and 160 mg/kg/day groups, respectively.

Clinical signs such as soiled perineal region, irregular respiration, subdued behavior and thin appearance were observed in 4, 4, 2 and 3 dams, respectively, at 160 mg/kg/day in late gestation period (GD 18- GD 21), these were considered dose related. A decrease in food consumption was observed at 160 mg/kg/day resulting in decreased mean body weight and decreased weight gain. In addition, gravid uterine weight, corrected terminal body weight and net body weight change were decreased compared to controls.

At caesarean section, a dose related change was observed in the number of dead fetuses at 160 mg/kg/day. An 8-fold increase in dead fetuses resulted in an increase (2.3-fold over controls) in post-implantation loss and a decrease (89.2% of controls) in live fetuses, and these changes were considered adverse. Mean fetal weight at 160 mg/kg/day group was 88.2% in both sexes (total value), 88.3 % in males and 88.9 % in females of the control values. A decrease in fetal weights of both sexes was not considered to be a direct effect of OBSH on fetuses, but secondary to maternal toxicities including the decrease in food consumption and subsequent body weight reduction.

External fetal examination did not reveal any abnormality. No visceral malformations was observed in any of the treatment groups. No skeletal malformations observed.

Incomplete ossification of interparietal and parietal bones and large fontanelle occurred at high incidence (2.08 %, 2.71 % and 4.58 %, respectively) in fetuses at 160 mg/kg/day, these skeletal variations were considered adverse. However, these were not thought to be caused by direct effects of OSBH on fetuses but indirectly by ossification retardation related to a marked reduction of fetal weights due to maternal toxicities.

In conclusion, clinical signs, a decrease in mean body weight, weight gains and food consumption were observed at 160 mg/kg/day. In addition, at caesarean section, an increase in the number of dead fetuses was observed at 160 mg/kg/day, and it resulted in an increase in post-implantation loss and a decrease in live fetuses. At the same dose level (160 mg/kg/day) of maternal effect, a decreased fetal weight and an increased incidence of skeletal variations related to retarded ossification such as incomplete ossification of interparietal and parietal bones and large fontanelle were observed considered to be secondary secundary to maternal toxicity. Therefore, under this study condition the maternal no-observed-adverse-effect level (NOAEL) is considered to be 60 mg/kg/day based on abnormal clinical signs and a decrease in body weight and food consumption at 160 mg/kg/day, and the NOAEL for prenatal development is 60 mg/kg/day based on increased number of dead fetuses, decreased fetal weights and retarded ossifications at 160 mg/kg/day.