Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From October 30, 2001 to March 25, 2002.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline test with GLP compliance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 440
GLP compliance:
yes
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
The test animals were juvenile female rats- animals recommended in 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 animals were transported together with foster dams from the supplier to Bayer AG. After the arrival, the animals intented for this study were acclimatized to conditions in the animal room for 3 days, until the commencement of the study. Their healthy situation was monitored during this period.
Only healthy animals showing no clinical signs were taken for the study. The rats were not vaccinated or dealt with anti-infectives either before receipt or during the acclimatization or treatment periods.
During the adaptation phase, the rats were conventionally kept in polycarbonate cages type III (one foster dam with six or seven inmature animals per cage). The cages were not changed during the acclimatization.
During the test period, the animals were conventionally kept in polycarbonate cages type III (three animals per cage). Low-dust wood shaving type BK 8/15 (supplier: Ssniff, Spezialdiäten GMBH, Soest/ Westphalia) were used as litter. The wood shavings were spot-checked for contaiminants levels and records are filed at Bayer AG. The analytical results afforded no evidence for an effect on the study objective.
Cages and bedding material were not changed during the test. The cages containing the experimental rats were palced on racks, separated by groups, in ascending animal number order. All animals taking part in the study were kept in the same animal room.

Administration / exposure

Route of administration:
subcutaneous
Vehicle:
corn oil
Details on exposure:
The dose levels were chosen on basis of the previous study (T9071164). In the study female rat was treated sub-cutaneously with 1000 mg/kg. No effects on the treatment area were observed.

Groups of 6 juvenile female rats of the strain HsdCpb:WU were administered the test substance once per day at levels of 0 (untreated), 0 (vehicle control), 200 and 1000 mg/kg body weight sub-cutaneously for a period of three days. Two additional groups of 6 juvenile female rats were set. The two groups were respectively treated with 0.3 and 1.0 μg/kg 17 α-Ethinylestradiol (positive control) once a day for 3 consecutive days.

In this study the dose schedule and the distribution of control animals and animals treated with the test substance by group are shown in table 1 in the section of "Any other information on materials and methods incl. tables"

Before administration, the animals were weighted, sorted according to weight, and randomly allocated to the individual groups. The randomization lists were generated by a computer program.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3 days
Frequency of treatment:
once per day
Duration of test:
4 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
200 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Each group contains 6 female immature rats. 6 groups were set in total, thus 36 rats were used totally.
Control animals:
yes, concurrent vehicle
Details on study design:
Environmetal conditions:
The environmental conditions in the animal room were standardized as follows:
Room temperature: 22±2 ℃
Relative humidity: approx. 55±5%
Light/dark cycle: 12-hour artificial lighting
air exchange rate: approx. 10 times per hour
Occasional deviations from these conditions occured, e.g. due to cleaning the animal room. They had no perceptible effect on the study routine.

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.
The tap water was of drinking water quality (in accordance with the German Drinking Water Standards "Trinkwasserverordnung" of December 5, 1990, Bundesgesetzblatt No. 66, part 1, issued on December 12, 1990, pages 2612-2629) and was supplied from polycarbonate bottles. The results of the water analyses were archived at BAYER AG. The available data did not indicate that the objective of the study had been influenced.

Inspection of experimental animals
The animals were inspected at least once a day. Any clinical signs and abnormalities were recorded. Body surfaces and orifices, posture, general behavior, breathing and excretory products were assessed. Findings and abnormalities were recorded both using a coding system and uncoded.
If animals get sick, they are set apart, observed more frequently and sacrificed prematurely, if death seems imminent.

Determination of feed consumption:
The feed intake of the rats was determined per group at the end of the study on the day 3. These primary data were used to calculate the means for 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.

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.

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 (pathology no. 6136). Due to the request of the sponsor, histopathological investigations were not performed.

Organ Weights
The following exsanguinated organs of the animals sacrificed at necropsy were weighted 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.
Statistics:
The results of the animal observations, organ, body and feed weights were collected and processed on-line and off-line.
The quatitative results for individual animals were used to calculate arithmetic group means and standard deviations. The results for the groups that received the test substance were compared with those for the vehicle control group. The vehicle control group was additionally compared with the untreated control group. Significant differences were indicated by "+" for p ≤ 0.05 and "++" for p ≤ 0.01.
The statistical evaluation of data related to body and organ weights as well as feed intake was performed by SAS® routines.
Statistical evaluation on body weight and organ weight data were processed by Dunnset test in connection with a variance analysis. Relative organ weights were submitted to a logarythmic transformation before the statistical analysis.

Results and discussion

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Oestrogenic/uterine increasing

Observed effects

Appearance and behavior:
No systemic clinical signs were observed;

Mortality
No animal died throughout the treatment period.

Feed consumption
No toxicologically relevant differences in mean feed consumption per animal/day, or per kg body weight/day were detected. The detailed results please refer to the table 2 in the section of "Any other information on results incl. tables".

Body weights
Comparing with the untreated control and vehicle control, there was no significant effect of the treatment group on body weight observed.

Findings at Necropsy
No pathological change has been observed at 1000 mg/kg of the test substance and below. However, the uteri were enlarged at 0.3 and 1.0 μg/kg 17α-Ethinylestradiol.

Oragn Weights
At 1000 mg/kg of the test substance and below no effect was observed on uterine weight. The positive controls gave the results the significantly higher uterine weights than the controls. Details please refer to the table 3 in the section of "Any other information on results incl. tables".

Any other information on results incl. tables

Table 2 Feed consumption

Feed consumption
Dose 3days
g/day/animal g/kg b.w./day
Females
0(untreated) 1.9 59
0(vehicle) 1.9 58
0.3μg/kg 17α-Ethinylestradiol 2.0 63
1.0μg/kg 17α-Ethinylestradiol 2.3 71
200 mg/kg test substance 2.1 67
1000 mg/kg test substance 1.8 56

Table 3 Organ weight

Dose Body weight absolute uterus weight Relative uterus weight
g mg mg/100g b. w.
wet blotted wet blotted
0(untreated) 32 33 27 102 83
0(vehicle) 33 32 27 97 83
0.3μg/kg 17α-Ethinylestradiol 32 59 ++ 53 ++ 184 ++ 166 ++
1.0μg/kg 17α-Ethinylestradiol 33 120 ++ 89 ++ 366 ++ 270 ++
200 mg/kg test substance 31 30 25 96 82
1000 mg/kg test substance 32 29 26 92 82

Applicant's summary and conclusion

Conclusions:
No oestrogenic effects were detected at doses of 1000 mg/kg of the test substance and below.
Executive summary:

Groups of immature female rats were administered the test substance once a day at levels of 0 (untreated), 0 (vehicle control), 200 and 1000 mg/kg body weight sub-cutaneously for a period of consecutive tree days. Besides two additional positive control groups containing 0.3 and 1.0 μg/kg 17α-Ethinylestradiol were set and treated same with testing groups. The vehicle used for all groups except untreated control was corn oil.

Regarding state of health and general behavior of the animals, there was no difference between the untreated and treated animals of all groups.

No treatment related mortality has been found.

No toxicologically significant effect on the feed intake was observed.

Body weights were not affected by treatment.

After necropsy, a dose-dependent enlargement of uteri and an increased uterine wet and blotted weight were observed in the positive controls. However, no effect on uteri was observed at either 200 or 1000 mg/kg test substance treated groups.