Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- public name of test material:
Reaction mass of 6,13-dichloro-3,10-bis{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino)ethyl]-amino}- polycarboheterocyclo 4,11-disulfonic acid, mono and/or disodium salt and 6,13-dichloro-3-{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino) ethyl]amino}-10-[(2-{[4-(ethenylsulfonyl)alkanoyl]amino}ethyl)amino] polycarboheterocyclo -4,11-disulfonic acid, mono and/or di sodium salt
- Physical state: solid, dark blue powder
- Analytical purity: approx. 86.6%
- Lot/batch No.: VER 2108 BOP 02/07
- Expiration date of the lot/batch: November 30, 2012
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Weight at study initiation: Females: 153.6-214.7g, (mean: 180.49 g, ± 20%= 36.10 g); Males: 298.4-320.7 g, (mean: 307.89 g, ± 20%= 61.58 g)
- Fasting period before study: No data
- Housing: The animals were housed individually in IVC cages (except during mating period when 2 females were paired with one male), type III H, polysulphone cages on Altromin saw fiber bedding (Lot No.: 101109, 291109, 301109 and 190110)
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice (Lot No.: 1130, 1552, 1323 and 0958)
- Water (e.g. ad libitum): Free access to tap water, acidified using sulfuric acid to a pH of approximately 2.8
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

IN-LIFE DATES: From: 28 Apr 2010 To: 28 Sept 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Sterile water (Manufacturer: Braun)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in sterile water. The vehicle was chosen based on the test item’s solubility. The test item formulation was prepared freshly on each administration day before the administration procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed on samples collected at at various intervals.
Samples for analysis of the dose formulations of the test item in the vehicle (nominal concentration) were taken in the first and last week of the study for all doses.
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. Samples were taken in the first and last week of the study.
All formulation samples were stored at -20 °C and analyzed after completion of the in-life phase of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 100380.
Details on mating procedure:
After acclimatisation, females were paired with males as per the ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to regularize the number of animals for terminal sacrifice on particular day. The subsequent morning and the next morning onwards, the vaginal smear of female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights were comparable with each other.
Duration of treatment / exposure:
gestation day 5-19
Frequency of treatment:
Daily
Duration of test:
Animals were sacrificed on gestation day 20.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 160, 400, 1000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
20 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: According to the results of the dose range finding study (BSL Study No. 100324).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed twice daily during the entire gestation period except during weekends and holidays where clinical observation was made only once. Mortality, morbidity, pertinent behavioural changes and all signs of overt toxicity were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the day of receipt to ensure that the body weights were within the + 20 % variation.
The sperm positive females were weighed during GD 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except on the day of receipt.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption of pregnant females was measured on GD 5, 8, 11, 14, 17 and 20. The food consumption was presented for period 0-5, 5-8, 8-11, 11-14, 14-17, and 17-20. The food consumption was measured neither for males during the entire study nor for both male and females during the mating period.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Examinations: At the time of termination, the presumed pregnant females were examined macroscopically for any structural abnormalities or pathological changes which might have influenced the pregnancy.
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
Fetal examinations:
- External examinations: Yes: All fetuses from particular dam were identified by different colored strings, weighed and sexed based on the anogenital distance. Each fetus was examined for external anomalies.
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one interval to the next.
For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control and test groups. Fetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Chi-square test.The statistical analysis was performed with GraphPad Prism (Version V) software (p<0.05 was considered as statistical significant).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical Observation:
Few major clinical signs observed were slightly reduced sponstaneous activity, piloerection, vocalisation, aelopecia and blue coloured feces. These various clinical signs in treatment and control group were not considered to be test item related.

Prenatal Data:
Statistically significant increase in group mean number of male fetuses in MD and HD group and sex ratio in HD group was observed when compared with controls. Group mean number of females was decreased in MD and HD group withought reaching statistical significance. This significant difference in various parameters was attributed to the gender imbalance and reason for this effect could not be established in this study.

Litter Data:
Statistically significant increase in group litter mean weight and male litter weight was observed in MD and HD when compared with controls. There was also decrease in female litter weight observed in HD when compared with controls. Group mean number of males was increased in MD and HD and group mean number of females was decreased in MD and HD as compared to controls. Total Number of fetuses remained unaffected in treated groups when compared with controls. This significant difference in various litter data parameters was attributed to the gender imbalance and reason for this effect could not be established in this study.

Gross Pathology:
The terminally sacrificed animals belonging to the control and treatment groups revealed lesions like kidney cyst, pale kidneys, lung with white spots. There was increased number of incidences of blue/green discolouration of various visceral organs observed in treated groups and which could be attributed to the blue colur of the test item and as such of no toxicological significance.
The pattern of gross pathological lesions at necropsy in very few animals from different treatment groups suggested that the lesions were of spontaneous/ incidental in nature.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Few gross external abnormalities were seen among the control and treatment groups. Predominant external finding noted was haematoma on various body locations in control and treated groups.

Skeletal examination of the Alizarin red stained fetuses revealed a range of abnormalities which were of a type or which occurred at an incidence comparable in treated groups when compared with controls. Statistically significant increase in incidence of incomplete ossification of 5th and 6th sternebra and significant decrease in incidences of ansent 4th metacarpal (bilateral) was observed in LD and HD resepectively. However, due to lack of dose dependency in this effect indicates no relevance with treatment. There was no indication of a compound related trend in the type and incidences of other abnormalities and they were therefore considered to be spontaneous in nature.

Internal observation of the fetal viscera by free hand microdissection technique revealed a range of visceral abnormalities in all groups including control. There was statistically significant decrease in kidney- hydronephrosis (Left) in LD and MD group. Since these visceral abnormalities observed in treated groups were infrequencies comparable or even less in numbers to controls, therefore no toxicological significance can be attributed to these findings and considered to be spontaneous in nature.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, the repeated dose administration of test material to pregnant female Wistar rats at dosages of 160, 400 and 1000 mg/kg body weight from gestation day 5 to 19 revealed no major toxicological findings in females and fetuses.
Based on the data generated from this study, The NOAEL for both maternal toxicity and fetal toxicity of test material in wistar rats was 1000 mg/kg body weight.
Executive summary:

This prenatal developmental toxicity study of test material was conducted in pregnant female wistar rats to detect the possible adverse effect on pregnant females and embryofetal development when administered by oral gavage from gestation day 5 to 19. Nulliparous and non pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of sperm positive vaginal smears (GD 0). Four groups of presumed pregnant females were dosed daily by oral gavage with 0, 160, 400 and 1000 mg/kg bw per day of test material at dose volume of 10 mL/kg body weight. Control animals were handled identically as treated groups and received sterile water as a vehicle in similar volume as treated groups.

The test item formulation was prepared freshly and dose volumes were adjusted based on the most recent body weight measurement.

Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured on various gestation days.

The treated and control females were sacrificed on respective gestation day 20. Followed by the gross necropsy evaluation of the females, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late) live and dead fetuses. Fetuses were identified by color strings, sexed and weighed. All fetuses were observed for external abnormalities, half of the fetuses for the visceral abnoramlities, craniofacial examination and remaing half of the litter for skeletal abnormalities.

Uteri of the non pregnant females were processed with 10% ammonium sulphide solution and checked for the early embryonic deaths if any.

No test item related clinical observations were observed in any of the dose group females during the entire period of the treatment when compared with controls. Two animals (51 and 73) from MD and 1 animal (94) from HD were died during the treatment period due to gavaging error.

Body weight, body weight change and food consumption remained unaffected throughout the treatment period.

Statistical analysis of prenatal data revealed no significant effect on prenatal parameters like terminal body weight, gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantations, live fetuses, dead fetuses, resorptions, percent preimplantation loss and post implantation loss in the treated groups when compared with controls. However, statistically significant increase in group mean number of male fetuses in MD and HD group and sex ratio in HD group was observed when compared with controls. Group mean number of females was decreased in MD and HD group withought reaching statistical significance.

Statistically significant increase in group litter mean weight and male litter weight was observed in MD and HD when compared with controls. There was also decrease in female litter weight observed in HD when compared with controls. Group mean number of males was increased in MD and HD and group mean number of females was decreased in MD and HD as compared to controls. Total number of fetuses remained unaffected in treated groups when compared with controls.

Decrease in pregnancy rate was observed in control group (73.08%), LD (80.00%), and MD (80.77%) as compared to HD (92.00%).

Few gross external abnormalities were seen among the control and treatment groups.

Skeletal examination of the Alizarin red stained fetuses revealed a range of abnormalities which were of a type or which occurred at an incidence comparable in treated groups when compared with controls. Statistically significant increase in incidence of incomplete ossification of 5th and 6th sternebra and significant decrease in incidences of ansent 4th metacarpal (bilateral) was observed in LD and HD resepectively.

Internal observation of the fetal viscera by free hand microdissection technique revealed a range of visceral abnormalities in all groups including control. There was statistically significant decrease in kidney- hydronephrosis (Left) in LD and MD group.

Craniofacial examination by razor blade serial sectioning technique revealed a range of visceral abnormalities in all groups including controls.

The terminally sacrificed animals belonging to the control and treatment groups revealed lesions like kidney cyst, pale kidneys, lung with white spots. There was increased number of incidences of blue/green discolouration of various visceral organs observed in treated groups. All these effects were not related to substance toxicity.

In conclusion, the repeated dose administration of test material to pregnant female Wistar rats at dosages of 160, 400 and 1000 mg/kg bw from gestation day 5 to 19 revealed no major toxicological findings in females and fetuses. Based on the data generated from this study, The NOAEL for both maternal toxicity and fetal toxicity of test material in wistar rats was 1000 mg/kg bw.