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Toxicological information

Toxicity to reproduction: other studies

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Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2, to November 8, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline test with GLP

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Validation work on in-vivo uterotrophic screening assay
Deviations:
not specified
Principles of method if other than guideline:
OECD, Protocol of the conduct of the rodent uterotrophic assay, Draft protocol B-Immature female rats with sub-cutaneous administration (April 20, 2000).
GLP compliance:
yes
Type of method:
in vivo

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl salicylate
EC Number:
204-263-4
EC Name:
2-ethylhexyl salicylate
Cas Number:
118-60-5
Molecular formula:
C15H22O3
IUPAC Name:
2-ethylhexyl 2-hydroxybenzoate
Test material form:
other: liquid
Details on test material:
chemical name: Benzoic acid, 2-hydroxy-, 2-ethylhexyl ester

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
The study was conducted on juvenile female rats - animals recommended 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 Winkelmarjin GmbH Experimental Animal Breeders in Borchen, District of Paderborn were used.
Animals of this strain have been used for many years at the BAYER AG for toxicological studies. Historical data on their physiology and spontaneous alterations is available. The state of health of the breed is monitored and the animals routinely spot-checked for the main specific pathogens. The results of these examination are filed at Bayer AG.
The animals were transported together with foster dams from the supplier 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, of health was monitored also during this period.
Only healthy animals showing no clinical signs were used for the study. They were not vaccinated or treated with anti-infectives either before receipt or during the acclimatization or treatment periods.
Housing condition:
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, Spezialdiaten GmbH, Soest/ Westphalia) were used as litter. The wood shavings were spot-checked for contaminants levels and the records are filled 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 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.
Identification of the Experimental Animals
The animals were identified using cage cards recording the test substance, animal numbers, dose, sex and study number as well as by individual marking using a saturated aqueous picric acid solution.
Cleaning, Disinfection,Pest Control
The animal room was cleaned once weekly and disinfected at least once a month with a 2.5% solution of Tego® 2000 provided from a centralized supply. At the same time it was ensured that the diet was not contaminated, and that there was no contact with the test animals. Cockroach traps were used for pest control. The racks were cleaned at regular intervals. Drinking bottles, cage lids containers were also replaced regularly. The entire cages were cleaned with hot water. A detergent was added to the final rinse, which was allowed to come into contact with the outside of the cages only.
Environmental Conditions
The environmental conditions in the animal room were standardized as foliow:
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/water: ad-libitum

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Groups of 6 juvenile female rats of the strain HsdCpb:WU were administered once a day at levels of 0 (untreated), 0 (vehicle control), 200 and 1000 mg/kg body weight sub-cutaneously for a period of three days. In two additional groups of 6 juvenile female rats each the females were treated once a day consecutive days with 0.3 and 1.0 µg/kg 17α-Ethinylestradiol (positive control). The vehicle used for all groups except untreated control was corn oil.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
not applicable
Duration of treatment / exposure:
3 days
Frequency of treatment:
once daily
Duration of test:
3 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (untreated), 0 (vehicle control), 200 and 1000 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
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.
Inspection of Experimental Animals
The experimental animals were inspected at least once a day. Any clinical sighs (findings) and abnormalities were recorded. Body surfaces and orifices, posture, general behavior, breathing and excretory products were assessed. Findings a 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.
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.
Determination of Body Weight
The body weights of the individual experimental animals were determined before 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. Due to the request of the sponsor, histopathological investigations were not performed.
Organ Weights
The following exsangulnated organs of the animals sacrificed at necropsy were 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.
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 group means and standard deviations. The results for the groups that received substance were compared with those for the vehicle control group. The control group was additionally compared to 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 is performed using SAS® routines.
Statistical evaluation on body weight and organ weight data were done using the Dunnet test in connection with a variance analysis. Relative organ weights submitted to a logarythmic transformation prior to 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: No uterine weight increase or other toxic effects

Observed effects

Regarding state of health or general behavior of the animals, there was no difference between the untreated and treated animals of all groups.
No treatment related mortality was observed.
No toxicologically significant effect on the feed intake was observed.
Body weights were not affected by treatment.
At necropsy, a dose-dependent enlargement of uteri and an increased u and blotted weight were observed at 0.3 and 1.0 pg/kg 17a-Ethinylestradiol, positive control). No effects on uteri were observed at 200 and 1000 mg/kg test article.

Applicant's summary and conclusion

Conclusions:
It is concluded that no oestrogenic effects were detected in the uterotrophic assay on juvenile female rats at subcutaneous doses of 1000 mg test article per kg body weight and below.
Executive summary:

The study objective was conducted to detect estrogenic effects of the test item in the uterotrophic assay following repeated sub-cutaneous adrrlinistrallion of the test substance to juvenile female Wistar rat (three-day treatment).

The study methodology conformed to the OECD Validation work on in-vivo uterotrophic screening assay, OECD, Protocol of the conduct of the rodent uterotrophic assay, Draft protocol B-Immature female rats with sub-cutaneous administration (April20, 2000).

Groups of 6 juvenile female rats of the strain HsdCpb:WU were administeredonce a day at levels of 0 (untreated), 0 (vehicle control), 200 and1000 mg/kg body weight sub-cutaneously for a period of three days. In two additional groups of 6 juvenile female rats each the females were treated once a day consecutive days with 0.3 and 1.0 µg/kg 17α-Ethinylestradiol (positive control). The vehicle used for all groups except untreated control was corn oil. No effects on uterine weight were observed at 200 and 1000 mg/kg test article. An enlargement of uterus was observed and increased uterine weights were determined at positive control.

In conclusion, no oestrogenic effects were detected in the uterotrophic assay onjuvenile female rats at sub-cutaneous doses of 1000 mg /kg body weight and below.