N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2-methoxy-5-methyl-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012 - 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 421) performed under GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Section 4, No. 421, “Reproduction/Developmental Toxicity Screening Test”adopted on 27 July 1995.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide

EC Number:

235-425-2

EC Name:

N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide

Cas Number:

12225-06-8

Molecular formula:

C32H24N6O5

IUPAC Name:

3-hydroxy-4-{[2-methoxy-5-(phenylcarbamoyl)phenyl]diazenyl}-N-(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species/strain: Crl: WI(Han): Wistar rats (Full-Barrier)
- Source: e.g. Charles River, 97633 Sulzfeld, Germany
- Sex: Males and non-pregnant nulliparous females
- Age at the study initiation: 10-11 weeks old
- Weight at study initiation: Body weight at the beginning of the study: interval within ± 20% of the mean weight.
The range of the body weight was:
Females: 169-217 g, (mean: 196.33 +/- 20%= 39.27 g)
Males: 260-294 g, (mean: 278.15 g, +/- 20%= 55.63 g)
- Housing: housed individually in IVC cages (except during the mating period when one female was paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (Lot No.261111)
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet: Altromin 1324 maintenance diet for rats and mice (Lot No.0715), ad libitum
- Water: ap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically), ad libitum
Certificates of food, water and bedding are filed at BSL BIOSERVICE.
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 55+/-10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The animals were dosed with the test item on 7 days per week for a period of approximately 54 days. The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28 days.

The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 5 mL / kg body weight.

For each animal, the individual dosing volume was calculated on the basis of the most recently measured body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), study week 3 (first week of mating), study week 5 (gestation) and study week 7 (gestation/lactation) - (total 16 samples)
Samples for homogeneity were taken from the top, middle and bottom of the high dose, medium dose and low dose preparation in study week 1 and 5 (total 18 samples).
All samples were analysed on the day of sampling within 6 hours after preparation.
All samples of dosing formulations were sent to Analytic department of BSL BIOSERVICE Scientific Laboratories GmbH on particular day of sampling.
Formulation analysis was performed in accordance with GLP.

Details on mating procedure:

Animal were paired in the ratio of 1:1 (male to female). The subsequent morning and the next morning there onwards the vaginal smear of the females were checked to confirm the evidence of mating. Day of vaginal plug and/or sperm occurrance was considered as day 0 of gestation.

Duration of treatment / exposure:

Males 28 days; Females: Approx. 54 days

Frequency of treatment:

7 days/week

Duration of test:

58 days

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Number and Sex of the Animals:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group). The study included three dose groups (LD, MD and HD) and one control group (C).

Preparation of the Animals:
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals were healthy and none of them shown any pathological signs before the first administration. Before the first administration, all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females. The animals were acclimatised for at least five days before the first dose administration.

Experimental group and Dosage:
In consultation with the sponsor and based on the a BSL dose range finding study, doses levels of 100, 300, 1000 were selected for the 3 dose groups (LD, MD and HD) and 1 control group (C).
Dose concentration was based on the purity/content (content C.I.) of the test item (97.3 %)
The animals in the control group were handled in an identical manner to the dose group subjects and received corn oil in the same volume as used for treatment groups.

Administration of Doses:
The animals were dosed with the test item on 7 days per week for a period of approximately 54 days. The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28 days.
The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 5 mL / kg body weight.
For each animal, the individual dosing volume was calculated on the basis of the most recently measured body weight.

Mating:
Animal were paired in the ratio of 1:1 (male to female). The subsequent morning and the next morning there onwards the vaginal smear of the females were checked to confirm the evidence of mating. Day of vaginal plug and/or sperm occurrence was considered as day 0 of gestation. Cages were arranged in such a way that possible effects due to cage placement was minimised.

Clinical Observation:
Animals were observed for clinical signs during the entire treatment period. General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily (and once daily during weekends and holidays) all animals were observed for morbidity and mortality. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded.


Body Weight and Food Consumption:
The body weight of all animals was recorded once before assignment to the experimental groups and on the first day of administration.
In the male animals, the body weight was taken weekly during the entire study period and on day of terminal sacrifice.
In the female animals the body weight were taken weekly during the pre-mating period, on gestation day 0, 7, 14, 20 and on post-natal day 0 (within 24 hours of parturition) and post-natal day 4 along with pups.

Food consumption was measured on the corresponding day of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period and post mating period in males.

Litter observations:
The duration of gestation was recorded and is calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing actual numbers on the back with the help of permanent marker In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Sperm Analysis:
At necropsy (one day after the last administration) one epididymis and one testis was separated and used for evaluation of sperm parameters. Epididymal sperm motility and testicular sperm count was evaluated in all male animals using Hamilton Thorn Sperm Analyser (TOX IVOS Version 13.0 C).

Gross Pathology:
Males were sacrificed after the completion of mating period (total dosing of 28 days) and females were sacrificed on respective post natal day 4 along with pups. At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
Pups sacrificed on day 4 post-partum were carefully examined for gross external abnormalities.
The number of implantation sites and corpora lutea was recorded in all pregnant females by gross observations.
The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole) were preserved in 10 % neutral buffered formalin. Testes and epididymides were initially preserved in modified Davidson’s Solution for approximately 24 hours and transferred to 10 % neutral buffered formalin.

Organ Weight:
The testes, epididymides, prostate and seminal vesicles with coagulating gland of all male adult animals and ovaries, uterus with cervix of all female adult animals were weighed.
Paired organs were weighed separately.

Histopathology:
A full histopathological evaluation (after the preparation of paraffin sections and haematoxylin-eosin staining) was carried out on all animals of the control and high dose groups which are sacrificed at the end of the treatment period.
Histopathological examinations were not extended to animals of the other dose groups, as no treatment-related changes were observed in the high dose group.
A detailed qualitative examination of the testes was made taking into account the tubular stages of the spermatogenic cycle at the evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
Gross lesions macroscopically identified were examined microscopically.
The histological processing of tissues to microscope slides were performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS- Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: prior to the assignment, daily during the study period

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: before assigmnet to study group, on day 1 of dosing, weekly during the study period, day before sacrifice

OTHER: Mating behaviour, organ weight, Gross pathology and Histopathology.

Ovaries and uterine content:

macropscopic and micropscopic examination.

Fetal examinations:

Number, weight and sex of pups, No. of still births, live births, runts and gross external abnormalities on PND 0 and 4.

Statistics:

A statistical assessment of the results of the body weight, food consumption, litter data and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.5.01 software (p<0.05 is considered as statistically significant).

Indices:

Copulation, Fertility, Delivery and Viability Indices

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality:
No mortalities were observed at any dose levels of male and female animals.

Clinical Observation:
No test item related clinical signs were observed in male and female animals.
Few spontaneous clinical signs observed occasionally in female animals were alopecia on various body parts (1/10 in Control and 4/10 in HD females), red nasal discharge (1/10 in LD females), slight piloerection (1/10 in LD, MD and HD females) and moderate piloerection (1/10 in HD females).

Body Weight and Body Weight Change:
In male no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups except statistically significant decrease in body weight change during premating day 1-7 in LD group when compared with controls. Due to lack of dose dependency, this significant effect on male body weight change was not considered to be treatment related.
In females, statistically significant decrease in mean body weight on day 7, 14 and decrease in body weight change during premating day 1-7 was observed in HD group when compared with controls. As this decrease in HD female body weight and body weight change was marginal and no effect on body weight development was observed during gestation and lactation period, this significant effect on female body weight was not considered to be toxicologically relevant.

Food Consumption:
In males and females, statistical analysis of food consumption data revealed no test item related effect on food consumption in treatment groups during entire study period when compared with controls.

Gross Pathology:
At necropsy of male (after minimum total dosing of 28 days - on day 29) and females (on post-natal day 4) by using a high dose of Ketamine/Xylazine (2:1), macroscopic examination of the animals revealed no test item related macroscopic findings in males and females. Few spontaneous gross pathological findings observed in male and female animals were Peyer´s patches - granulated (1/10 in C males), mesenteric lymph nodes - discoloured red (1/10 in C and LD, 2/10 and 5/10 in MD and HD males), adrenal glands - discoloured pale (2/10 in C and 1/10 in MD and HD males), ileum/Peyer´s patches - discoloured red (2/10 in MD and 1/10 in LD and HD males), ileum - discoloured red (5/10 in HD and 2/10 in LD and MD males), yellow spot on both epididymides (2/10 in HD males) and caecum, colon and rectum- gased (1/10 in MD females).
These gross pathological findings were spontaneous in nature and as such not a systemic effect due to the test item administration.

Organ Weight:
In males and females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology
At terminal sacrifice, macroscopic organ findings were few and none of them was considered to be test item-related. No test item-related histological findings were noted in the male and female reproductive organs.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

other: content C.I.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

other: content C.I.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Soft tissue and skeletal examinations were not made.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Dose Formulation Analysis:

Concentration analysis of formulation samples was determined in study week 1, 3, 5, and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 96.9%, 98.9%, and 100.7% of the nominal concentration, respectively.

Homogeneity of formulation samples was determined in study week 1 and 5 for all dose groups. The mean recoveries observed for LD group were 98.0% and 106.9%, for MD group 98.9% and 96.0%, and for HD group 100.0% and 94.8% of the nominal value. The coefficients of variation of the different sampling locations (top, middle, bottom) were in LD group 1.3% and 0.2%, in MD group 1.0% and 1.8%, and in HD group 1.2% and 0.4%.

Applicant's summary and conclusion

Conclusions:

In conclusion, the repeated dose administration of the test item to the male (28 days) and female (maximum 54 days) Wistar rats at dosages of 0, 100, 300 and 1000 mg/kg body weight revealed neither mortalities nor findings of toxicological relevance.
Based on the data generated from this reproduction/ developmental toxicity screening test with the test item, the no observed adverse effect level (NOAEL) is believed to be 1000 mg/kg body weight for reproduction/ developmental toxicity screening in male and female rats.

Executive summary:

The aim of this study was to assess the possible effect of the test item on male, female fertility and embryofetal development in Wistar rat. This study was also aimed to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

In this study, four groups comprised of 10 adult males and 10 non pregnant nulliparous female rats (Wistar Crl:WI) were dosed daily by oral gavage with 100, 300 and 1000 mg/kg body weight per day of test item at dose volume of 5 mL/kg body weight. The test item was formulated in corn oil. Control animals were handled identically as treated groups and received corn oil in similar volume as treated groups.

The doses evaluated were 0, 100, 300, 1000 mg/kg.

Dose concentration was based on the purity/content (content C.I.) of the test item (97.3 %).

The test item formulation was prepared freshly and administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for a total period of 28 days. Dose volumes were adjusted weekly based on the recent body weight measurement.

Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured weekly except during the mating period and post mating period in males where food consumption was not measured.

After 14 days of premating treatment to both male and female, animals were paired (1:1) for 14 days. The subsequent morning onwards, the vaginal smears of females were checked to confirm the evidence of mating in the form of sperm positive vaginal smears. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters were weighed within 24 hours of parturition and on day 4 post-partum.

Males and females were sacrificed on treatment day 29 and post natal day 4 respectively and subjected to necropsy. Non pregnant females (47 and 77) were sacrificed on respective day 26 after the evidence of mating. The wet weight of male and female reproductive organs was taken and preserved in 10 % neutral buffered formalin except testes and epididymides which were initially fixed in Modified Davidson’s fixativefor approximately 24 hours before they were transferred to 10% neutral buffered formalin. Histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically.

Summary Results

Clinical Signs and Mortality:

No test item related clinical signs and mortalities were observed in both males and females.

Body Weight Development

In male no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups except statistically significant decrease in body weight change during premating day 1-7 in LD group when compared with controls.

In females, statistically significant decrease in mean body weight on day 7, 14 and decrease in body weight change during premating day 1-7 was observed in HD group when compared with controls.

Effect on body weight or body weight change was not considered to be toxicologically relevant.

Food Consumption:

In males and females, statistical analysis of food consumption data revealed no test item related effect on food consumption in treatment groups during entire study period when compared with controls.

Litter Weight Data:

Group mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 remained unaffected in all treated groups when compared with controls.

Precoital Interval and Duration of Gestation:

No treatment related effect was observed on precoital interval and duration of gestation and values were comparable between the groups. All pregnancies resulted in normal births.

Pre and Post Natal Data:

Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls.

Litter Data:

No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4. All group mean and individual values for various litter data parameters from treatment groups were comparable with the controls.

Reproductive Indices:

No treatment related effect on copulation index, delivery index, fertility index and viability index was observed when compared with controls.

Pup Survival Data:

Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups and no pup mortality was observed in this study. However, 2 pups from HD group female 80 (pup No. 1 and 2) went missing on PND 1 and those were presumed to be cannibalized by the dam.

Pup External Findings:

No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, few spontaneous findings were observed which were not considered to be test item related.

Sperm Analysis:

Statistical analysis of sperm motility and testicular sperm head count data revealed no test item related effect on sperm parameters and all group mean and individual values from treatment groups were comparable with the controls.

Gross Pathology:

At necropsy, macroscopic examination of the animals revealed no test item related macroscopic findings in males and females. Few spontaneous gross pathological findings were observed in male and female animals and as such not a systemic effect due to the test item administration.

Organ Weight:

In males and females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology:

No treatment-related macroscopic organ findings were noted in this study, and histopathological evaluation of reproductive organs in control and high dose animals did not reveal any treatment-related changes.

Dose Formulation Analysis:

Concentration analysis of formulation samples was determined in study week 1, 3, 5, and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 96.9%, 98.9%, and 100.7% of the nominal concentration, respectively.

Homogeneity of formulation samples was determined in study week 1 and 5 for all dose groups. The mean recoveries observed for LD group were 98.0% and 106.9%, for MD group 98.9% and 96.0%, and for HD group 100.0% and 94.8% of the nominal value. The coefficients of variation of the different sampling locations (top, middle, bottom) were in LD group 1.3% and 0.2%, in MD group 1.0% and 1.8%, and in HD group 1.2% and 0.4%.

Stability in the vehicle:

 > 72 h at a concentration of 0.1 mg/mL and 50 mg/mL at room temperature (analysed by the sponsor)

Based on the data generated from this reproduction/ developmental toxicity screening test with the test item, the no observed adverse effect level (NOAEL) is 1000 mg/kg body weight for reproduction/ developmental toxicity screening in male and female rats.