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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-04-14 to 1993-05-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
Cited as Directive 87/302/EEC, part B, p.24
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Uvinul N 539
- Analytical purity: 98.3 %
- Lot/batch No.: 505396-70182 (2070182)
- Stability under test conditions: confirmed
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
sexually mature, virgin Wistar rats (Chbb :THOM (SPF))
- Source: Karl THOMAE, Biberach an der Riss, FRG
- Age at study initiation: 61-70 days, upon arrival at test facility; 70-80 days (start of the study on day 0 of gestation)
- Weight at study initiation: mean weight ca. 228.2 g (day 0 of gestation)
- Fasting period before study: no data
- Housing: individually in type DK III stainless steel wire mesh cages (floor area about 800 cm² )
- Diet (e.g. ad libitum): ground Kliba 343 feed rat/mouse/hamster, Klingentalmühle AG, Kaiseraugst, Switzerland
- Water (ad libitum): tap water from bottles
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1993-04-05 (arrival) To: 1993-05-03 (sacrifice)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions were prepared twice during the administration period because the stability of the test substance solutions over a period of at least 7 days had been verified analytically. For the preparation of the solutions, an appropriate amount of the test substance was weighed, subsequently topped up with olive oil DAB 10 and intensively stirred.

VEHICLE
olive oil, DAB 10
- Concentration in vehicle: 20, 80, 200 mg/ml for the low, mid and high dose level, respectively
- Amount of vehicle (if gavage): 5 ml/kg bw/d
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of oily solutions of the test substance for a period of at least 7 days were carried out for an acute oral toxicity study (Project No.: 10A0227/921093).
Samples of the test substance solutions were sent to the analytical department of BASF Aktiengesellschaft twice during the study period for verification of the concentrations.
The test substance solutions were analyzed by photometry.
As the test substance preparations were true solutions, the homogeneity had not to be proven analytically.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight , ca. 15-16 h
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
day 6 through day 15 of gestation
Frequency of treatment:
daily
Duration of test:
until day 20 of gestation
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 400, 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
25 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Since Uvinul N 539 is absorbed through the gastrointestinal tract, oral administration of Uvinul N 539 (by gavage) was selected as the route of choice for this prenatal toxicity study.
The selection of doses for the present examination was based on the results of a preceding study in which Uvinul N 539 was applied to 5 male and 5 (virgin) female Wistar rats (Chbb :THOM SPF) in olive oil at a dose of 2000 mg/kg body weight for 15 consecutive days. As a control, 5 male and 5 female rats were treated with the vehicle only. The following findings were obtained:
Mortality and body weight data were not affected. In the second week of the study, salivation was observed each time after administration of the test substance. The blood sampling at the end of the study revealed statistically significantly increased serum-gamma-glutamyltransferase (SGGT) and cholesterol values in treated males and females; in treated males creatinine, total protein, and globulins were increased, in the 2000 mg/kg females red blood cells, hemoglobin, and hematocrit were decreased. From the above mentioned changes in clinical chemistry and hematology, however, only the changes in SGGT and cholesterol were assessed as being treatment-related. The urine of the animals was discolored (slightly red). Absolute and relative liver weights were increased, more pronounced in the females than in the males.
Taking into consideration the results of the above mentioned study with administration of Uvinul N 539 the following doses were fixed for the full-scale prenatal toxicity study in rats with Uvinul N 539:
100 mg/kg body weight: as the expected no observed adverse effect level
400 mg/kg body weight: as the intermediate dose
1000 mg/kg body weight: at the high dose level at which overt signs of maternal toxicity were not expected; testing at higher dose levels, however, was not deemed to be necessary due to the recommendations given in the test guidelines concerning the "Limit Test".

- Rationale for animal assignment: random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once per day

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 of gestation
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 pc. minus weight of the uterus before it was opened minus body weight on day 6 pc).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
With the exception of day 0, the consumption of food was determined on the same days as was body weight.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus, ovaries, liver

On day 20 pc, the dams were sacrificed in randomized order by cervical dislocation and the fetuses dissected from the uterus. After the dams had been sacrificed, they were necropsied and assessed by gross pathology. The uterus, the ovaries and the liver were removed and the following data were recorded:
- Weight of liver
- Weight of uterus before it was opened
- Number of corpora lutea
- Number and distribution of implantation sites classified as :
• live fetuses
• dead implantations:
a) early resorptions (only decidual or placental tissues visible or according to Salewski from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy)
b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c} dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

At necropsy each fetus was weighed, sexed and examined macroscopically for any external findings. The sex was determined by observing the distance between the anus and the base of the genital tubercle and was later confirmed in all fetuses fixed in BOUIN'S solution by internal examination. If there were discrepancies between the "external" and the "internal" sex of a fetus, the fetus was finally sexed according to the appearance of its gonads.
Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes and fluids were examined. Individual placental weights were recorded.
After these examinations, approximately one half of the fetuses per dam was placed in ethyl alcohol and the other half was placed in BOUIN's solution for fixation and further evaluation.

Soft tissue examination of the fetuses
After fixation in BOUIN's solution, approximately one half of the fetuses of the dams of all groups was examined for any findings in the organs. After these examinations the relevant fetuses were discarded.

Skeletal examination of the fetuses
After fixation in ethyl alcohol, the skeletons of approximately one half of the fetuses were stained. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After these examinations the relevant fetuses were retained by litter.
Statistics:
The data were evaluated statistically using the computer systems of the Department of Toxicology of BASF Aktiengesellschaft.
DUNNETT's Test was used for statistical evaluation of food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), liver weights (absolute and relative), weight of the uterus before it was opened, weight of fetuses, weight of placentae, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses.
FISHER's Exact Test was used for statistical evaluation of conception rate, mortality (of the dams) and all fetal findings.
Significance levels: p< 0.05, p< 0.01.
Historical control data:
yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
Clinical examinations
There were no mortalities in any of the groups.
Substance-induced salivation, which occurred shortly after the daily treatment and which disappeared - in most of the affected females - after ca. 6 hours latest, was observed in several dams of the 1000 mg/kg group, predominantly during the second half of the treatment period. Immediately after the last day of the treatment period (day 15 pc), salivation was not found any more in any of the substance-treated females. Moreover, some dams of the high dose group showed a transient reddish-brown discoloration of the fur in the anogenital region or urine-smeared fur on some days of the
treatment period. The reddish-brown discoloration of the anogenital region was probably caused by urine, because slightly red urine, even not observed in this study, was noted in a previous range-finding study with Uvinul N 539 at 2000 mg/kg bw/d. This discoloration, probably by the test substance itself or one of its metabolites, showed the systemic availability of the substance.
There were no differences of biological relevance between treated and control groups concerning
- mean food consumption
- body weight data (mean body weight, mean body weight change, corrected body weight gain
- uterus weight
- conception rate
- mean number of corpora lutea
- mean number of implantation sites
- pre- and post implantation losses
- number or resorptions
- number of viable fetuses
All values are within the range of biological variation.

Absolute and relative liver weights were slightly, but statistically significantly higher in the 1000 mg/kg group (approx. 9%) than in the control group . Furthermore, the relative liver weight was marginally, but still statistically significantly increased (approx. 6%) in test group 2 (400 mg/kg body weight/day). These effects are related to the test substance administered.
At necropsy single animals of all groups including the controls showed lungs with edema; this finding, which showed no relation to dosing, has to be related to the sacrifice of the animals. Moreover for one dam of test group 1 (100 mg/kg body weight/day) enlarged spleen and white foci in the liver were recorded (possibly due to an unspecific, spontaneously occurring inflammation).

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
There were no differences of biological relevance between treated and control groups concerning
- sex ratio
- mean weight of placentae
- mean weight of fetuses
All values are within the range of biological variation.
The incidence and type of external, soft tissue, and skeletal malformations/variations/unclassified observations were within range of historical control data.
The administration of Uvinul N 539 to pregnant rats did not increase the overall malformation rate; the few malformations recorded are assessed as being of spontaneous nature. All of it are also present at a low incidence in the historical control data and/or most malformations appeared at substantially similar incidences even in the actual control group. Moreover, no uniform trend within the malformation pattern was evident.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: no test substance related effects observed

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion