2-phenyl-1H-benzimidazole-5-sulphonic acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from October 30, 2001 to March 19, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD, Protocol of the conduct of the rodent uterotrophic assay, Draft protocol B - Immature female rats with sub-cutaneous administration (April 20, 2000).

Principles of method if other than guideline:

Test article were sub-cutaneously administered once per day on three consecutive days from the start of the study until either spontaneous death or scheduled sacrifice of the animals on fourth day. Clinical observations were performed daily. Body weights were determined daily. Feed intake was determined per group at termination (day 3). Gross necropsy(with uterus weights and tissue sampling) was performed on all animals at termination.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-phenyl-1H-benzimidazole-5-sulphonic acid

EC Number:

248-502-0

EC Name:

2-phenyl-1H-benzimidazole-5-sulphonic acid

Cas Number:

27503-81-7

Molecular formula:

C13H10N2O3S

IUPAC Name:

2-phenyl-1H-benzimidazole-5-sulphonic acid

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Experimental Animals:
The study was conducted on juvenile female rats - animals recommend in the OECD protocol for these validation studies. At the start of the study, the animals were 19 days old and had starting weights of 29 - 36 g.
SPF-bred Wistar rats of the strain HsdCpb: WU from the Harlan Winkelmann GmbH Experimental Animal Breeders in Borchen, District of Paderborn were used.The state of health of the breed is monitored and the animals spot-checked for the main specific pathogens.
The animals were transported together with foster dams from the supplier to Bayer AG. After the arrival, the animals intended for this study were acclimatized to conditions in the animal room for 3 days, until the start of the treatment. Their state of health was monitored also during this period.
Only healthy animals showing no clinical signs were used for the study. The animals were not vaccinated or treated with anti-infectives either before receipt or during toe acclimatization or treatment periods.

Housing conditions:
During the adaptation period, the animals were conventionally kept in polycarbonate cages type III (one foster dam with six or seven juvenile animals per cage). The cages were not changed during the adaptation period.
During the test period, the animals were conventionally kept in polycarbonate cages type III (three animals per cage), Low-dust wood shavings type BK 8/15 supplier: Ssniff, Spezialdiäten GmbH, Soest/ Westphalia) were used as litter. Tie wood shavings were spot-checked for contaminants levels and the records are filed at Bayer AG. The analytical results afforded no evidence for an effect on study objective.
Cages and bedding material were not changed during the test period.
The cages containing the experimental animals were placed on racks, separated by groups, in ascending animal number order. All animals taking part in this study were kept in the same animal room.
The environmental conditions in the animal room were standardized as follows:
room temperature: 22 ± 2 °C
relative humidity: approx. 55 ± 5 %
light/dark cycle: 12-hour artificial lighting
air exchange rate: approx. 10 times per hour
Diet:The animals were given "NAFAG No. 9441 Long Life W 10" (manufacturer: Eberle Nafag AG, Gossau - CH) and tap water (drinking bottles). Food and water were available for ad-libitum consumption.

Administration / exposure

Route of administration:

subcutaneous

Vehicle:

corn oil

Details on exposure:

Test substance were sub-cutaneously administered once per day on three consecutive days from the start of the study until either spontaneous death or scheduled sacrifice of the animals on fourth day.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

three consecutive days

Frequency of treatment:

once per day

Details on study schedule:

Dosage Form, Doses, Study Groups
The test substance, the vehicle control, and the positive control were sub-cutaneously administered once per day on three consecutive days from the start of the study until either spontaneous death or scheduled sacrifice of the animals on fourth day.
The dose levels were chosen on basis of discussions with the sponsor and on basis of the results of a pilot study (T9071164), In this study one female rat each was treated sub-cutaneously with 1000, 500, or 200 mg/kg. At 200 mg/kg and above on the treatment area skin changes were observed, and at 500 mg/kg additionally loss of hair.
In this study not only the test item but additionally four other test compounds were investigated and compared to the same control groups. The results for each test compound will be reported separately. The dose schedule and the distribution of the control animals and animals treated with the test item by group are shown in the following table:

Group no dose No of animals Animal no.
1 0 (untreated) 6 1-6
2 0 (vehicle) 6 7-12
3 0.3 µg/kg EE 6 13-18
4 1.0 µg/kg EE 6 19-24
5 50 mg/kg test group 6 25-30
6 200 mg/kg test group 6 31-36

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day

No. of animals per sex per dose:

6

Control animals:

other: untreated and vehicle(group 1 and 2)

Details on study design:

Before administration, the animals were weighed, sorted according to weight, and randomly allocated to the individual groups. The randomization lists were created with a computer program.

Positive control:

17a-Ethinylestradiol (Sigma, Lot 057H1178)

Examinations

Parental animals: Observations and examinations:

1, General investigations
1.1.Inspection of Experimental Animals
The experimental animals were inspected at least once a day. Any clinical signs (findings) and abnormalities were recorded. Body surfaces and orifices, pos general behavior, breathing and excretory products were assessed. Findings abnormalities were recorded either using a coding system or else uncoded.
If animals become ill, they are set apart, observed more frequently and sacrificed prematurely, if death seems imminent.
1.2. Determination of Feed Consumption
The feed intake of the rats was determined per group at the end of the study on day 3. These primary data were then used to calculate the means for the feeding period, the consumption per animal/day, per group/day and per kg body weight per day. The algorithm used for calculating intake of feed is described in the Annex of the report.
1.3. Determination of Body Weight
The body weights of the individual experimental animals were determined before the beginning of the study and daily thereafter up to scheduled necropsy.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

no data

Postmortem examinations (parental animals):

1. Necropsy
The animals were necropsied after exsanguination under deep ether anesthesia. Uterus and vagina of all animals were fixed in 10% neutral buffered formalin.
The fixed material was retained. Due to the request of the sponsor, histopathological investigations were not performed.
2.Organ Weights
The following exsanguinated organs of the animals sacrificed at necropsy weighed in the unfixed state:
Uterus (wet and blotted).
The uterine weights are specified in both absolute and relative terms. The relative weights were calculated by normalization to 100 g body weight (individual organ weight divided by body weight times 100). The body weight used as a basis for this calculation was determined immediately prior to necropsy of the pertinent animal.

Postmortem examinations (offspring):

no data

Statistics:

The results of the animal observations, organ, body and feed weights were collected and processed on-line and off-line.
The quantitative results for individual animals were used to calculate arithmetic grThe statistical evaluation of data related to body and organ weights as well as feed intake is performed using SAS® routines.
Statistical evaluation on body weight and organ weight data are done using the Dunnet test in connection with a variance analysis. Relative organ weights are submitted to a logarythmic transformation prior to the statistical analysis.

In case of numbers of values too low to calculate test statistics this is indicated by ‘o’ or ’-’ in the tables shown in the results section and by ‘nc’ (not calculated) or ‘0’ in the tables of the Annex. The individual results listed in the Annex to this report have been rounded off. Calculation of means and variances was based in part on the non-rounded original values. Some of the values in the tables of individual body weights may be missing. This may be the consequence of a technical defect which occurred during on-line data collection or of a value which can not be listed (because of an error in weighing and a failure to meet printer format) or is unrealistic account of a weighing error and deleted.

Reproductive indices:

no data

Offspring viability indices:

no data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

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:

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, non-treatment-related

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:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Details on results (P0)

No systemic clinical signs were observed. No animal died throughout the entire treatment period.
No toxicologically relevant differences in mean feed consumption per animal/day, or per kg body weight/day were detected.
No significant effect of treatment on body weights were observed.
No pathological changes were observed at 200 mg/kg bw/day test article and below. The uteri were enlarged at 0.3 and 1.0 µg/kg 17a-Ethinylestradiol.
At 200 mg/kg bw/day test article and below no effect were observed on uterine on the uterine weight.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Remarks:

dose received by subcutaneous injection

Sex:

female

Basis for effect level:

other: At the dose tested, no effect on uterus weight (i.e. endocrine effect) and no effect on body weight, feed consumption or signs of toxicity were observed.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

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:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, no oestrogenic effects were detected at doses of 200 mg/kg bw/day of test article and below.

Executive summary:

This study was conducted to detect estrogenic effects of the test item conformed to the OECD Validation work on in-vivo uterotrophic screening assay following repeated sub-cutaneous administration. Groups of 6 juvenile female rats of the strain HsdCpb:WU were administered test substance once a day at levels of 0 (untreated), 0 (vehicle control), 50 and 200 mg/kg body weight/day sub-cutaneously for a period of three days. Regarding state of health or general behavior of the animals, there was no difference between the untreated and treated animals of all groups. No toxicologically significant effect on feed intake, body weight and organ weight was observed.