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Administrative data

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: second stage of the validation of the OECD rodent uterotrophic assay (GLP, QAU)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: The aim of the study was to obtain initial data for the uterotrophic assay within the OECD validation project and based on the "Protocol for the Conduct of the OECD Rodent Uterotrophic Assay", Draft Protocol A of April 21, 2000
GLP compliance:
yes (incl. certificate)
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): dibutylphthalate
No additional details available

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: the dams and the pups (Wistar, CRL : WI (GLX/BRL/HAN) IGS BR) were supplied on July 25, 2000 (Supplier : Charles River Laboratories, Germany). The litters were standardized to 8(preferably female) pups/litter in order to have the same conditions for all animals. If there, were fewer than 8 female pups in one litter, male pups were supplemented to obtain the number of pups required. The pups had an age of 12 days (day of birth = day 0)
- Age at study initiation: at an age of 18 days the pups were weaned. Thereafter, the female pups were assigned to the different test groups using a randomization program, according to their weight one day before the beginning of the treatment period. The selected sexually immature females had an age of 19 days on study day 0 (first day of treatment)
- Weight at study initiation: body weight range, 31 .8 - 39.7 g; mean of means, 35.47 g; +/- 5%, 33.70 - 37.24 g; mean min., 35.27 g; mean max., 35.68 g
- Housing: the animals were housed in Makrolon cages (type M II, No. of animals per cage: 3)
- Diet (e.g. ad libitum): ground Kliba feed rat/mouse/hamster (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): tap water
- Acclimation period: the dams and the pups were allowed to acclimatize for at least 7 days prior to dosing. At an age of 18 days the pups were weaned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Remarks:
Olive oil EP/DAB
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): the preparations were carried out once in olive oil. For this purpose, the coded vials which had been weighed and coded by the sponsor were dissolved or dissolved partially in ethanol (about 99.8%), the amount of ethanol not to exceed 10 vol.% of the final volume. Subsequently, the preparations were topped up to their maximum volume (see table below) and stirred. The vessels were jacketed with aluminium foil to avoid the incidence of bright light.
- Storage temperature of food: the preparations were stored in a refrigerator.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No analyses of the test substance and the reference estrogen were carried out within the OECD validation project with the exception of the test substance characterization by the manufacturer.
Duration of treatment / exposure:
3 consecutive days
Frequency of treatment:
once a day
Doses / concentrations
Remarks:
Doses / Concentrations:
0 and 1000 mg/kg bw
Basis:
actual ingested
a dose volume of 5 ml/kg body weight/day was used
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
- A dose volume of 5 ml/kg body weight/day was used. The calculation of the volume administered to each female was based on the individual body weight identified daily before the doses were administered.
- The carrier control group, consisting of 6 females, was dosed with the vehicle only (olive oil), in the same manner as the test substance treated animals.

Examinations

Examinations:
MORTALITY
Viability was checked twice daily.

BODY WEIGHT
Body weights of the animals were recorded daily before the daily intubations throughout the study period.

CLINICAL SIGNS
The state of health of the animals was checked each day.

NECROPSY AND UTERI WEIGHT
- The animals were anesthetized with CO2 and sacrificed by decapitation about 24 hours after the last substance administration on study day 3.

- The abdominal wall was opened in the region of the pubic symphysis. The uteri were detached from the abdominal wall together with the ovaries. Urinary bladder and ureters were removed ventrally and laterally from the uterus and vagina. Fibrous adhesions between the rectum and the vagina were detached until the junction of vaginal orifice and perineal skin was identified. The sectioning for removal was made caudal to the cervix.
After the uteri had been thoroughly dissected free from the surrounding adipose tissue, the ovaries were detached. At necropsy, care was taken that any existing fluid in the uterus was not getting lost before weighing. After the ovaries had been removed, the uteri were transferred into petri dishes. Subsequently, each uterus was weighed with luminal fluid (wet weight) and without luminal fluid (blotted weight). For blotting, both uterus horns were pierced with the tip of a scalpel, topped with a dry filter paper (Schleicher & Schüll) and gently pressed to absorb the luminal fluid with a covered bale of cellulose. Thereafter, the uteri were fixed in 4% formaldehyde solution to be available for possible histopathological examination in the future.
Positive control:
The test substance was tested together with 17-Ethinyl estradiol (CAS 57-63-6; 0.001 and 0.003 mg/kg per day); methoxychlor (CAS 72-43-5; 300 mg/kg per day); genistein (CAS 446-72-0; 300 mg/kg per day); 2,4'-DDT (CAS 789-02-6; 300 mg/kg per day); bisphenol A (CAS 80-05-7; 600 mg/kg per day); and nonylphenol (CAS 104-40-5; 250 mg/kg per day)

Results and discussion

Details on results:
CLINICAL OBSERVATIONS AND MORTALITY
None of the female rats, which received the test substance orally at 1000 mg/kg body weight/day showed abnormal clinical signs or any disturbances of the general behavior during the administration period. Moreover, there were no unscheduled mortalities.

BODY WEIGHT (See Table 1)
Mean body weights and mean body weight gains were not influenced by the oral administration of 1000 mg test substance/kg body weight/day. All differences between the test substance - treated rats and the vehicle control group concerning mean body weight data are without any biological relevance. They did not attain statistical significance.

UTERUS WEIGHT (See Table 1)
Mean absolute and relative uterus weights (wet and blotted) of the animals receiving 1000 mg test substance/kg body weight/day were not influenced by the oral administration and were similar to the concurrent control values.

Any other information on results incl. tables

Table 1: Mean body and uterine weights

 

 

Parameter

Test group (dose in mg/kg per fay)

Olive oil

17-Ethinyl estradiol (0.001)

17-Ethinyl estradiol (0.003)

Dibutylphthalate (1000)

Methoxychlor (300)

Genistein (300)

2,4’-DDT (300)

Bisphenol A (600)

Nonyphenol (250)

 

Body weight (g)

Day 0

34.2±2.36

33.1±3.04

33.3±3.16

34.8±2.51

34.5±2.94

34.4±2.56

36.3±2.43

34.3±2.81

34.6±2.88

Day 1

36.7±2.52

36.5±3.81

35.2±2.79

36.3±3.18

36.0±3.47

36.4±3.21

36.2±2.28

36.4±2.81

31.8±3.68*

Day 2

39.3±3.13

39.6±3.65

39.6±2.34

39.8±2.76

37.7±4.28

38.8±5.26

36.1±3.85

39.3±3.04

29.4±1.25*

Day 3

40.8±4.67

42.3±4.22

41.7±2.10

42.5±2.94

39.7±4.81

41.0±6.62

37.1±5.61

42.9±3.14

26.0±1.27*

Uterine weight (g or %)

Absolute wet

0.031±0.006

0.103±0.0273*

0.159±0.0745*

0.032±0.007

0.094±0.0187*

0.080±0.0065*

0.184±0.0687*

0.043±0.0044*

0.061±0.0071

Relative wet

0.077±0.011

0.248±0.0867*

0.379±0.1639*

0.076±0.171

0.239±0.0493*

0.200±0.0424*

0.512±0.2144*

0.099±0.0066*

0.234±0.0157

Absolute blotted

0.030±0.006

0.084±0.0123*

0.111±0.0206*

0.030±0.006

0.089±0.0157*

0.076±0.0061*

0.108±0.0123*

0.041±0.0044*

0.049±0.0007

Relative blotted

0.073±0.01

0.202±0.045*

0.266±0.0456*

0.071±0.0155

0.255±0.0418*

0.191±0.0416*

0.296±0.0494*

0.095±0.0062*

0.191±0.0121

Applicant's summary and conclusion