Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-11-18 to 1996-12-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study reliable without restrictions Deviations from the current guidelines OECD 414 (2001) and EU method B.31 (2008) - according to the guideline, normally the test substance should be administered daily from implantation (e.g., day 5 post mating) to the day prior to scheduled caesarean section. In this study the dosing of the animals was stopped five days before the caesarean section. - according to the guideline, the test substance should be administered at approximately the same time each day. It was not stated in the study report, if this was done. - according to the guideline, females should be killed one day prior to the expected day of delivery. Normally the rat has a gestation period of 22 days. The rats in this study were killed on day 20 of gestation. - according to the guideline, the gravid uteri including the cervix should be weighed. In this study, the uteri and the cervix were not weighed, but the placenta. - according to the guideline, particular attention should be paid to the reproductive tract of the foetus during examination of the foetus. This appears not to have been done during the study. - according to the guideline, the justification of choice of vehicle should be given. The justification was missing in the study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
1988
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. deficiencies" above
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
1981-05-12
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. deficiencies" above
GLP compliance:
yes (incl. certificate)
Remarks:
signed 1996-02-27
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- CAS name: 1H-Benzimidazole-4,6-disulfonic acid-2,2´-(1,4-diphenylene) bis, disodium salt
- Molecular formula: C20H12N4O12S4Na2
- Molecular weight: 674g/mol
- physical state: solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Weight at study initiation ( after mating at allocation to treatment group): 214 to 272 g (females)
- Age at study initiation: ten to twelve weeks (females)
- Housing: upon arrival, the females were housed in groups of five in polypropylene cages with stainless steel grid floors and lids, suspended over paper-lined polypropylene trays. During the mating period females were transferred to a similar type cage with a sexually mature male on a maximum of 2 female: 1 male basis.
Following evidence of successful mating, the animals were caged individually in polypropylene cages with solid floors and stainless steel grid tops. Softwood chips were used as bedding material.
- Diet (ad libitum): pelleted diet (SQC Rat and Mouse Breeder Diet No.3 Expanded, Special Diets Services Limited, Witham, Essex, UK)
- Water (ad libitum): water
- Acclimation period: at least 13 days before mating

ENVIRONMENTAL CONDITIONS
- Temperature range: 19 - 23 ° C
- Relative humidity range: 40 - 70%
- Air changes: at least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was suspended daily in 1% carboxymethyl cellulose. For each dose level, an appropriate aliquot of test material was weighed into a glass jar end vehicle was added to make the calculated final volume. Homogeneity was assured by mixing the formulations with a Silverson mixer/homogeniser.
Formulations were prepared daily and used immediately after preparation.
Dose volume: 10 mL/kg
The dose administered was adjusted for bodyweight during the dosing period.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. Results show the formulations to be stable for at least two hours. Samples were taken of each test material formulation on three days during the study. On each day samples were taken immediately, 30 minutes and 60 minutes after sample preparation. The results indicate that the prepared formulations were within ± 8% of the nominal concentration.

For the results of the analysis please also see Section 8 Analytical methods (Entry: s_Wood & Dolemann_1997)
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: females were housed with males on 2 female : 1 male basis.
- Length of cohabitation and proof of pregnancy: the following morning each tray was checked for the presence of ejected copulation plugs. All females were checked for copulation plugs in situ, and then a vaginal smear was taken to check for the presence of sperm. Females showing evidence of sperm within the vaginal smear were separated from the male and designated Day 0 of gestation. These females were replaced with previously unmated females.
- The total mating period for this study was 6 days.
Duration of treatment / exposure:
Day 6 to Day 15 of gestation, inclusive
Frequency of treatment:
once daily
Duration of test:
20 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 400, 800 and 1200 mg/kg (dose concentration 0, 40, 80 and 120 mg/mL)
Basis:
other: actual administered
No. of animals per sex per dose:
24 female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected following a preliminary study. During the preliminary study, the test material was administered orally by gavage to mated females once daily from Day 6 to Day 15 of gestation, inclusive (Day 0 of gestation was evidence of sperm within vaginal smear). The dose levels were 400, 800 and 1200 mg test substance/kg (dose concentration: 0, 40, 80 and 120 mg/mL respectively; dose volume 10 mL/kg). A concurrent control group was dosed with the vehicle (1% carboxymethyl cellulose) only. In each dose group were 8 mated female rats.
Results:
- There were no deaths during the study.
- No clinical signs were noted during the study.
- There were no effects on bodyweight; bodyweight gain was comparable for all groups throughout the study period.
- Food consumption was comparable for all groups throughout the study period.
- At 1200 mg/kg/day, one animal was found to have stenosis of the right uterine horn proximal to the cervix at terminal necropsy. No other abnormalities were noted at necropsy.
- The pregnancy rate was 8/8, 7/8, 7/8, 7/8 for 0, 400, 800 and 1200 mg/kg respectively. There were no treatment-related effects on uterine/implantation data.
- There were no treatment-related foetal findings.
Treatment of pregnant females with the substance at dose levels up to 1200 mg/kg/day produced no evidence of maternal or foetal toxicity. There were no treatment-related effects on pregancy or foetuses at any dose level. The results from this study suggest that a top dose level of up to 1200 mg/kg/day should be used for the main study, taking into account the limit dosage for substances of low toxicity.
Taking into account the limit dosage given in the respective guidelines a top dose level of 1200 mg/kg/day was chosen after consultation with the Sponsor.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Morbidity/Mortality: all females were checked twice daily during the normal working week and once daily at weekends.
Clinical observations: all females were observed once daily, in the morning throughout gestation and additionally, one hour after dosing, throughout the dosing period, for clinical signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: all females were weighed on Day 0, Day 3, Day 6 to Day 9, Day 12, Day 15, Day 18 and Day 20 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for individual animals was recorded for discrete periods throughout the study on Day 0 to 3, Day 3 to 6, Day 6 to 9, Day 9 to 12, Day 12 to 15, Day 15 to 18 and Day 18 to 20 of gestation.

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20; all surviving females were killed by carbon dioxide asphyxiation. Each animal was examined externally and internally for macroscopic abnormalities.
- Organs examined: the ovaries and uteri of pregnant females were removed. The placenta was weighed.
- The liver and kidneys of all adults were weighed at necropsy.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No, only the placenta was weighed.
- Number of corpora lutea: Yes
- Number of implantations: Yes; number, position and type of intrauterine implantation
- Number of early resorptions: Yes; no visible distinction between placental/decidual tissue and embryonic tissue.
- Number of late resorptions: Yes; separate embryonic/foetal and placental tissue visible. A foetus that had died shortly before necropsy. These were included as late deaths for reporting purposes.

All implantations and viable foetuses were numbered according to their intrauterine position as follows:
Left horn
L1 V1
L2 V2
L3 V3
L4 V4
L5 V5
L6 V6
L7 V7
L8 V8

Cervix

Right horn
R1 V9
R2 V10
R3 V11
R4 V12
R5 V13
R6 V14
R7 V15
R8 V16
V = viable foetuses
Fetal examinations:
The foetuses were killed by intraperitoneal injection of sodium pentobarbitone.

- External examinations: Yes; external foetal appearance, foetal sex, foetal weight
- Soft tissue examinations: Yes; alternate foetuses were identified using an indelible marker and placed in Bouin's fixative. After a minimum of 7 days, foetuses were transferred to 90% industrial methylated spirits (IMS) in distilled water and examined for visceral anomalies under a low power binocular microscope (Wilson, J.G. ( 1965). Embryological considerations in Teratology. in Teratology: Prinicples and Techniques, eds J.G. Wilson and J. Warkany, University of Chicago Press, Chicago, Illinois, pp 262 -277.).
- Skeletal examinations: Yes; the remaining foetuses were identified using colour coded wires and placed in 70% IMS in distilled water. The foetuses were eviscerated, processed and the skeletons stained with alizarin red (Dawson, A.B. (1926). A note on the staining of the skeleton of cleared skeletal specimens with Alizarin Red S. Stain Technol. 1, 123 -4.). The foetuses were examined for skeletal development and anomalies.
- Head examinations: No
Statistics:
Data were processed to give litter mean values, group mean values and standard deviations.
Bodyweight change: absolute bodyweight change of females during gestation was calculated and presented relative to Day 6 of gestation (Day 1 of treatment)
Statistical evaluation: The following parameters were analysed statistically, where appropriate using the test methods outlined below:
Female bodyweight change (relative to Day 6 of gestation), food consumption and organ weights: Levene's test for homogeneity of variance and one way analysis of variance with the Bonferroni multiple comparison test.
All caesarian necropsy parameters, foetal parameters including skeletal development, group incidence of specific visceral and skeletal anomalies: Kruskall-Wallis non parametric analysis of variance followed by pairwise analysis of control values against treated values using the Mann-Whitney 'U' test.
Indices:
Pre and post implantation loss:
% pre-implantation loss: [(number of corpora lutea - number of implantations) / number of corpora lutea] X 100
% post implantation loss: [(number of implantations - number of live foetuses) / number of implantations] X 100
Historical control data:
Background control data for rat teratology studies was provided for the following investigations:
- skeletal development
- foetal skeletal findings
- foetal external and visceral findings

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 data: there were no mortalities throughout the course of the study
- Clinical observations: there were no clinical signs of toxicity during either the dosing or off-dose periods of the study.
- Bodyweight: bodyweight gain for all dose groups was comparable to controls throughout the course of the study.
- Food consumption: there were no significant differences in food consumption during the course of the study for treated groups when compared to controls.
- Adult necropsy data: there were no significant differences in macroscopic necropsy findings observed for all groups. The findings observed were considered incidental.
- There were no significant differences in maternal liver and kidney weight.
- Uterine/implantation: for 0, 400, 800 and 1200 mg/kg dose groups there were 22/24, 23/24, 23/24 and 22/24 pregnant females.
- There were no significant treatment related differences in the uterine implantation data reported.
- The pre- and post-implantation losses were within acceptable limits for all dose groups and controls.
- The live litter size was similar for all groups.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
> 1 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
> 1 200 mg/kg bw/day (actual dose received)
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:
- There were no significant intergroup differences in mean foetal or placental weights and total litter weights were comparable for all groups.
- The type and incidence of foetal external findings showed no treatment related trends. All findings are commonly observed in this study type.
- For all dose groups and controls, there were no significant structural malformations.
- At visceral evaluation, the proportion of foetuses showing visceral changes were lower in all dose groups compared to controls. There were no treatment related differences in the distribution of findings throughout dose groups and controls.
- The degree of skeletal development was comparable for all groups including controls. The proportion of foetuses with variances in skeletal findings were comparable for all dose groups and controls. The type and distribution of skeletal variances amongst offspring from treated females showed no treatment related differences when compared to controls.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 1 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
At dose levels up to and including 1200 mg test substance per bodyweight there was no evidence of adult toxicity as determined by evaluation of maternal bodyweight change and food consumption throughout gestation. There were no clinical signs of toxicity during the dosing period and no significant macroscopic changes at caesarian necropsy. At these dose levels there was no evidence of embryotoxicity or foetotoxicity from the parameters evaluated at caesarian necropsy. Detailed foetal evaluation for differences in visceral findings showed no significant differences in the proportion of affected foetuses or distribution of the types of anomalies recorded. Evaluation of skeletal development showed no differences in the degree of skeletal development or in the proportion and distribution of skeletal variances recorded.
Females dosed with the substance from Day 6 to 15 of gestation at dose levels up to and including 1200 mg/kg bodyweight did not show evidence of maternal toxicity and there was no evidence of effects on the growth and development of offspring.