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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:
1992-02-21 - 1992-10-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methylpyrazole
EC Number:
215-925-7
EC Name:
3-methylpyrazole
Cas Number:
1453-58-3
Molecular formula:
C4H6N2
IUPAC Name:
3-methyl-1H-pyrazole
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Sexually mature, virgin Wistar rats (Chbb:TH0M (SPF)) supplied by Karl THOMAE, Biberach an der Riss, FRG, which were free from clinical signs of disease, were used for the investigations. This strain was selected since extensive experience is available on Wistar rats and this strain has been proved to be sensitive to substances with a teratogenic potential. Arrival of the animals on Feb. 24, 1992.
During the study period, the rats were housed singly in type DK III stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, FRG (floor area about 800 cm2) .
The cages with the test animals were arranged on the racks in such a way that uniform experimental condi¬tions (ventilation and light) were ensured.
The animals were accommodated in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20 - 24°C and a range of rela¬tive humidity of 30 - 70%. There were no deviations from these limits.
The day/night rhythm was 12 hours (12 hours light from 6.00 to 18.00 hours and 12 hours darkness from 18.00 to 6.00 hours).
Before the study started, the room was completely dis¬infected using a disinfection apparatus ("AUTEX” fully automatic final disinfecting apparatus using formalde¬hyde and ammonia). In general, each week the walls and the floor were cleaned with water containing about 0.5% Mikro-Quat (supplied by SCHULKE & MAYR GmbH).
The food used was ground Kliba 343 feed rat/mouse/ hamster supplied by KLINGENTALMUHLE AG, CH-4303 Kai- seraugst, Switzerland, which was available to the animals ad libitum throughout the study (from the day of supply to the day of necropsy), as was drinking water of tap water quality.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test substance was administered to the animals orally (by gavage) once a day during the period of major organogenesis (day 6 to day 15 p.c.). The volume administered each day was 10 ml/kg body weight. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 6 p.c.).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the aqueous test substance solutions over a period of
3 hours were carried out during the study. Furthermore, samples of the test substance solutions were sent to the analytical laboratories of BASF Aktiengesellschaft twice during and - in addition to times scheduled in the study protocol - also after the end of the study period for verification of the concentrations.*

The test substance solutions were analyzed by GC.
Details on mating procedure:
Mating took place from about 16.00 hours to about 7.30 hours on the following day. If sperm were detected microscopically in the vaginal smear in the morning, the animals were considered to be fertilized. This day was designated "day 0" (beginning of the study) and the following day "day 1" post coitum (p.c.).
Duration of treatment / exposure:
The test substance was administered to the animals orally (by gavage) once a day during the period of major organogenesis (day 6 to day 15 p.c.).
Frequency of treatment:
once daily
Duration of test:
20 days post coitum)
Doses / concentrationsopen allclose all
Dose / conc.:
90 mg/kg bw/day (nominal)
Dose / conc.:
45 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 pregnant female rats per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The female rats were about 56 - 63 days old when supplied. After an acclimatization period of at least 5 days, 2 - 4 untreated female rats were mated with one untreated fertile male animal of the same breed.
Mating took place from about 16.00 hours to about 7.30 hours on the following day. If sperm were detected microscopically in the vaginal smear in the morning, the animals were considered to be fertilized. This day was designated "day 0" (beginning of the study) and the following day "day 1" post coitum (p.c.).
At the beginning of the study (day 0, detection of sperm), the rats were 67 - 79 days old. Their mean weight was approx. 224.5 g.
On day 0, the animals were assigned to the different test groups according to a randomization plan (2).
The test substance was administered to the animals orally (by gavage) once a day during the period of major organogenesis (day 6 to day 15 p.c.). The volume administered each day was 10 ml/kg body weight. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 6 p.c.).
On day 20 p.c., all surviving animals were sacrificed in a randomized order. The fetuses were dissected from the uterus and further investigated with different methods.
Each day the aqueous test substance solutions were freshly prepared shortly before the test substance was administered. For the preparation of the solutions, an appropriate amount of the test substance was weighed, subsequently topped up with doubly distilled water and intensively stirred.

Examinations

Maternal examinations:
Food consumption, body weight, body weight gain , clinical symptoms, mortality, preimplantation loss, postimplantation loss, gross pathology
Ovaries and uterine content:
Weight of uterus before it was opened,
number and distribution of implantation sites classified as : live fetuses, dead implantations (early resorptions, late resorptions, dead fetuses)
Fetal examinations:
Macroscopic examination, determination of sex, viability, condition of the placentae, examination of the umbilical cords, the fetal membranes and fluids, placental weights, soft tissue examination of the fetuses, skeletal examination of the fetuses

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of the 45 and 90 mg/kg dams were lower in comparison to the relevant control values (45 mg/kg: day 8 p.c.; 90 mg/kg: days 8 - 20 p.c.). Weight gains of these dams were clearly impaired during the first days of the treatment period (45 mg/kg: days 6-8 p.c.; 90 mg/kg: days 6 - 10 p.c.). This corresponds to the reduced food consumption of the dams of these test groups . In the subsequent time, however, weight gains of the intermediate and high dose dams reached or even exceeded control values.
Body weights and body weight gains of the dams of test group 1 (15 mg/kg body weight/day) were similar to those of the controls . All observable differences between these two groups are without any biological relevance.
The corrected body weight gain (terminal body weight on day 20 p.c. minus weight of the uterus before it was opened minus body weight on day 6 p.c.) was statistically significantly reduced in test group 3 (90 mg/kg body weight/day), which has to be assessed as a substance-related effect. All other differences between the groups are without any biological relevance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Only one spontaneous finding was recorded for one ani¬mal (No. 37} of test group 1 (15 mg/kg body weight/ day), which showed a hydrometra and did not become pregnant.The uterus weight in test group 3 (90 mg/kg body weight/day) was statistically significantly reduced (about 15% lower than the corresponding control value), which is being assessed as a substance-related effect. All other differences between the groups are without biological relevance.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The conception rate varied between 88% and 100% (45 and 90 mg / kg groups).
There were no substance-related and/or statistically significant differences of biological relevance between the groups in conception rate, in the mean number of corpora lutea and implantation sites, in the values calculated for the pre- and the postimplantation losses or in the number of resorptions and viable fetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age .
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Dose group: 90 mg/kg
-reduced food consumption
-significantly lower body weights

Dose group: 45 mg/kg
-reduced food consumption

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean fetal body weights were statistically significantly lower in the intermediate and high dose groups (45 or 90 mg/kg body weight/day) when compared to the control values. This is a clear substance-induced effect. Mean fetal body weights in test group 1 (15 mg/kg body weight/day) were not influenced by the test substance administration
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1 - 3 (15, 45 and 90 mg/kg body weight/day) was comparable with the control fetuses. The differences observed in comparison to the control are without any biological relevance.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External examination of the fetuses
An external malformation was recorded for one intermediate dose fetus, only. For this fetus, a cleft palate was noted. This malformation is also present in the historical control data at a low frequency [see Volume III (Supplement)]. Therefore, this malformation is finally assessed as being of spontaneous origin.
The external examination of the fetuses revealed no variations in any group.
One so-called unclassified observation (placentae fused) was recorded for one fetus of test group 0 (control) and one fetus of test group 2 (45 mg/kg body weight/day)
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Various malformations of the sternum (sternebra(e) bipartite, ossification centers dislocated) and/or the vertebral column (thoracic vertebral body/bodies dumb¬bell-shaped (asym.) or bipartite (asym.) were seen in all groups including the control fetuses; furthermore, one control fetus had multiple malformations of the ribs and the vertebral column. While the fetal and litter incidence of the observed skeletal malformations is similar in the control and test groups 1 and 2 (15 or 45 mg/kg body weight/day), asymmetrically bipartite or dumbbel1 - shaped thoracic vertebral body/bodies occurred statistically significantly more often in the high dose fetuses. The relevant values for fetal and litter incidence in the 90 mg/kg group are considerably outside the historical control range.
The increased ocurrence of skeletal malformations of the thoracic vertebral column in the high dose group (90 mg/kg body weight/day) has to be attributed to the test substance administration.
The variations elicited were related to the ribs (shortened or absent 13th, accessory 14th, rudimentary cervical or wavy rib(s)) and the sternum (sternebra(e) of irregular shape or bipartite) and were found in all groups without a clear dose-response relationship. The statistically significantly increased occurrence of shortened 13th rib(s) in the high dose fetuses is with¬out any biological relevance; the relevant values are fully within the historical control range.In all groups signs of retardations (incomplete or missing ossification of vertebral bodies/arches, sternebra(e), metatarsalia, skull and/or the hyoid bone) were found. In the 90 mg/kg group, the number of fetuses with symmetrically dumbbel1 - shaped thoracic vertebral body/bodies, not ossified, incompletely ossified or "reduced in size" sternebra(e) and the overall number of fetuses with skeletal retardations is statistically significantly increased. Although some, but not all of these findings are still in the historical control range , the increased incidence of skeletal retardations is finally assessed as a substance-induced effect, because it has to.be seen in conjunction with the substance- related, statistically significantly lower fetal body weights in this group.
The fetal and litter incidences of skeletal retarda¬tions in test groups 1 and 2 (15 or 45 mg/kg body weight/day), however, are similar to that of the control group and do not show any relation to dosing.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The examination of the organs of the fetuses revealed severe malformations of the urogenital tract and/or the cardiovascular system in a total of 14 out of 163 fetuses (in 8 out of 25 litters) of the high dose group (90 mg/kg body weight/day). There were, however, no fetuses with soft tissue malformations in any of the other test groups (0, 15 or 45 mg/kg body weight/day).
Concerning the malformations of the urogenital tract, agenesia of the left kidney and the left ureter occurred in two fetuses of different litters, one of these fetuses showing in addition agenesia of the left uterine horn. For 5 high dose fetuses massive dilata¬tion of the urinary tract (renal pelvis and ureters) was recorded; particularly the ureters had up to about 4 times the size of those of the control. Another fetus of this group had an undescended left testis and showed an abnormal position of the left kidney.
A displaced aortic arch occurred in 6 fetuses of 2 litters and a globular shaped heart with dilatation of both ventricles was seen in two other fetuses (from two different litters).
With the exception of the latter finding (globular¬shaped heart) none of the malformations described before appear in the large historical control data base of the investigating laboratory [see Volume III (Supplement)] and are therefore assessed as clear substance-induced findings.
Variations [dilated renal pelvis and/or hydroureter (this term is used by the evaluating laboratory for slight to moderate, commonly seen dilatations of ureter(s))] were detected in all groups without any statistically significant differences between the groups. All values are fully within the range of biological variation.
No so-called unclassified observations (like blood coagulum around the bladder) were recorded.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Dose group 90 mg/kg:
-reduced fetal body weights
-delayed ossification
-malformations of the urogenital tract, cardiovascular system, thoracic vertebral bodies

Dose group: 45 mg/kg
-significantly lower fetal body weights
-no teratogenic effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
skeletal: vertebra
visceral/soft tissue: urinary
visceral/soft tissue: cardiovascular
Description (incidence and severity):
Various malformations of the sternum (sternebra(e) bipartite, ossification centers dislocated) and/or the vertebral column (thoracic vertebral body/bodies dumb¬bell-shaped (asym.) or bipartite (asym.) were seen in all groups including the control fetuses; furthermore, one control fetus had multiple malformations of the ribs and the vertebral column. While the fetal and litter incidence of the observed skeletal malformations is similar in the control and test groups 1 and 2 (15 or 45 mg/kg body weight/day), asymmetrically bipartite or dumbbel1 - shaped thoracic vertebral body/bodies occurred statistically significantly more often in the high dose fetuses. The relevant values for fetal and litter incidence in the 90 mg/kg group are considerably outside the historical control range.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
45 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Applicant's summary and conclusion

Conclusions:
Under the conditions of the described test, the no observable adverse effect level (NOAEL) on the maternal and fetal organism was 15 mg/kg body weight/ day.
Executive summary:

3 - METHYL-PYRAZOL was tested for its prenatal toxicity in Wistar rats. The test substance was administered as an aqueous solution to 22 - 25 pregnant female rats/- group by stomach tube in doses of 15, 45 and 90 mg/kg body weight on day 6 through day 15 post coitum. A standard dose volume of 10 ml/kg body weight was used. The control group was dosed with the vehicle only (doubly distilled water).


Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.


On day 20 post coitum, all females were sacrificed and assessed by gross pathology. The fetuses were dissected from the uterus, sexed, weighed and further investigated for any external, soft tissue and/or skeletal findings.


3 -Methyl-pyrazol caused marked signs of maternal toxicity at 90 mg/kg body weight/day and was still clearly toxic to the dams at 45 mg/kg body weight/day; 15 mg/kg body weight/day were tolerated by the dams without any adverse substance-related effects. Signs of embryo-/fetotoxicity, including clear indications for teratogenicity, occurred at the highest dose level; besides reduced fetal body weights and signs for a delayed ossification of the fetal skeletons, malformations of the urogenital tract, the cardiovascular system and the thoracic vertebral bodies were seen in a considerable number of high dose fetuses. In the 45 mg/kg fetuses embryo-/fetotoxic effects, but no indications for any substance-related teratogenic effect were observed, whereas no signs of developmental toxicity were found at the dose of 15 mg/kg body weight/day.