N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)benzenesulphonamide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Minor deviation in histopathology processing and clinical bochemistry parameters; while these deviations did not influence the quality or integrity of the present study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Specification of study design for extended one generation reproduction toxicity study with justifications:

- Premating exposure duration for parental (P0) animals : 14 days
- Termination time for F2 : Pups were sacrificed on post natal day 4.
- Route of administration : Oral gavage

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)benzenesulphonamide

EC Number:

274-040-4

EC Name:

N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)benzenesulphonamide

Cas Number:

69563-51-5

Molecular formula:

C21H16N2O5S

IUPAC Name:

N-(4-amino-3-methoxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)benzenesulfonamide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Q30450ADZX
- Expiration date of the lot/batch: 08/08/2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Solubility of the test substance in the solvent/vehicle: 3.26 mg/l in water

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9-10 weeks
- Weight at study initiation: Male: 253 - 281 g and Female: 171 - 191 g
- Housing:
Animals were housed in groups of 2 animals/ sex/ cage in IVC cages (type III H, polysulphone cages) during the premating period in both males and females and during postmating period in males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period and males were returned to its original cage. Each cage was provided with Altromin saw fibre bedding (lot no. 131113)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55±10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the evidence of mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimized.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28-29 days were completed. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study.

Frequency of treatment:

Daily up to 54 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw (total dose)

Remarks:

Low Dose

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw (total dose)

Remarks:

Medium dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

High dose

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28-29 days were completed. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. The control group was shared with BSL study 140271. The following doses were evaluated:
Control (C): 0 mg/kg/d
Low Dose (LD): 100 mg/kg/d
Medium Dose (MD): 300 mg/kg/d
High Dose (HD): 1000 mg/kg/d
The test item formulation was prepared once in every ten days and stored at 2-8°C. The test item was suspended in corn oil and dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight. During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals. Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five males and five randomly selected females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males and females from each group. Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment and at the end of the study. After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery 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 weighed within 24 hours of parturition and on day 4 post-partum. The males were sacrificed after completion of the mating period on treatment days 29 and 30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females (females 42 and 71) were sacrificed on day 26 from day of sperm positive vaginal smear or from the last day of mating period. Pups sacrificed on post natal day 4 and those found dead, were carefully examined for gross external abnormalities. A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups as well as all gross lesions from all groups, were examined by light microscopy. Furthermore, testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix and vagina were examined in all animals, and, on the testes, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Examinations

Parental animals: Observations and examinations:

The duration of the gestation was recorded and was calculated from day 0 of the 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 tattoo mark on paw. In addition to the observations of parent animals, any abnormal behavior of the offspring was recorded.

Litter observations:

The duration of the gestation was recorded and was calculated from day 0 of the 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 tattoo mark on paw. In addition to the observations of parent animals, any abnormal behavior of the offspring was recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: After mating (Days 29 and 30)
- Maternal animals: females along with their pups were sacrificed on post natal day 4. Non-pregnant females (females 42 and 71) were sacrificed on day 26 from day of sperm positive vaginal smear or from the last day of mating period.

GROSS NECROPSY
- Pups sacrificed on post natal day 4 and those found dead, were carefully examined for gross external abnormalities.

HISTOPATHOLOGY / ORGAN WEIGHTS
A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups as well as all gross lesions from all groups, were examined by light microscopy. Furthermore, testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix and vagina were examined in all animals, and, on the testes, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. Because of possible treatment-related findings noted in the high dose group, spleen and bone marrow (sternum) from 5 selected male and female animals of the low and medium dose groups were examined to establish a no-effect level.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

There were no effects on the duration of precoital interval and the duration of gestation in dose group animals when compared to the controls. There were no effects on the pre and post natal data including the no. of corpora lutea, no. of implantation sites, no. of live pups (PND 0 and 4), percent pre and post implantation loss. There were no effects on the copulation, fertility and delivery indices of dose groups when compared to controls. A fertility index (number of pregnant females/ number of copulated females X 100) observed in the control and the dose groups were C 90%, LD 100 %, MD 100% and HD 100 %. There was no effect on viability index of dose groups when compared to control. There were no effects of toxicological relevance on the litter data including no. of pups born, no. of live pups, no. of still birth and no. of runts on PND 0 and no. of male and female pups and sex ratio on PND 0 and PND 4. There were no effects of toxicological relevance on litter weight data including pup mean weight, total litter weight, male litter weight and female litter weight measured on PND 0 and PND 4. No effects on the survival of the pups from PND 0 to PND 4 wereobserved in any dose group when compared to controls. There were no gross external findings of pups of toxicological relevance in any of the dose groups when compared to the controls.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36014/W, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg/d for reproduction/ developmental toxicity.

Executive summary:

On the basis of this reproduction/developmental toxicity screening test with FAT 36034/W in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d conducted according to OECD 422 guideline (GLP-compliant), there were no effect on the reproduction/ developmental parameters. Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36014/W, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg/d for for reproduction/ developmental toxicity.