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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:
From Oct 2001 to Sep 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed according to OECD Guideline 414 and GLP.

Data source

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

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)
Remarks:
BASF SE, 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Imidazole
EC Number:
206-019-2
EC Name:
Imidazole
Cas Number:
288-32-4
Molecular formula:
C3H4N2
IUPAC Name:
1H-imidazole
Details on test material:
Imidazole (CAS: 288-32-4), purity 99.8%

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
SPECIES AND STRAIN
Time-mated Wistar rats (CrIGIxBrIHan:WI ) supplied by Charles River Laboratories, Germany
Animals free from clinical signs of disease, were used for the investigations.
The female animals were supplied at an age of about 70 - 84 days on October 09, 10 and 11, 2001.
Based on the pregnant animals the body weight on day 0 varied between 139.4 -10 186.4 g.

ANIMAL IDENTIFICATION
The animals were mated by the breeder and supplied on day 0 post coitum (= detection of vaginal plug/sperm).
They were assigned to the test groups by taken random selection from the transport box.
After randomization the rats were identified uniquely by ear tattoo.

REASON FOR SPECIES SELECTION
This strain was selected since extensive experience is available on Wistar rats and this strain has been proved to be
sensitive to substances with ateratogenic potential.

HOUSING AND DIET
The rats were housed singly from day 0- 20 p.c. in type DK III stainless steel wire mesh cages.

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 relative humidity of 30 - 70%. There were no deviations from these limits.

The day/ night rhythm was 12 hours (12 hours light from 6.00 a.m. to 6.00 p.m. and 12 hours
darkness from 6 .00 p.m. to 6.00 a.m.).
Before the study started, the animal room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalin-ammonia - based terminal disinfection). In general, each week the walls and the floor were cleaned with water containing about 0 .5% Mikro-Quat (supplied by ECOSAN GmbH, FRG).

The food used was ground Kliba maintenance diet rat/mouse/hamster meal, supplied by PROVIMI KLIBA SA, Kaiseraugst, Switzerland.
Foodwas 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 from water bottles.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
TS dissolved in doubly distilled water and the oral route was selected, since this has proven to be suitable for the detection of a toxicologicalhazard.
The test substance solutions were administered to the animals orally (by gavage) once a day from implantation to one day prior to the expected day of parturition (day 6 to day 19 p.c.) always at approx. the same time of day (in the morning). The animals of the control group were treated in the same way with the vehicle (doubly distilled water). The volume administered each day was 10 ml/kg body weight. The calculation of the volume administered was based on the last individual body weight. The fetuses were removed from the uterus and further investigated with different methods. Due to technical reasons, the study was carried out in 3 sections. Each dose group was represented in each section. A treatment interval of one day elapsed before the next section.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in doubly distilled water at room temperature were carried out with GC method before andafter the study had been initiated. Samples of the test substance solutions were sent to the analytical laboratory twice during the
study period (at the beginning and towards the end) for verification of the stability and/or the concentrations. Since the test substance preparations were true solutions, investigations concerning homogeneity were not necessary.
Details on mating procedure:
The animals were mated by the breeder ("time-mated") and supplied on day 0 post coiturn (= detection of vaginal plug / sperm).
The animals arrived on the same day (i .e. day 0 p.c.) at the experimental laboratory.
The following day was designed "day 1" post coitum (p.c.). Between start of the study (beginning of the experimental phase) and
first administration (day 6 p.c.) the animals were acclimated to the laboratory conditions.
Duration of treatment / exposure:
TS was administered by oral gavage once a day from implantation to one day prior to expected parturition, i.e. d 6-19 p.c.


Frequency of treatment:
7 d/wk
Duration of test:
Start of the experimental phase (day 0 p.c.) was on Oct 09, 2001; last sacrifice (day 20 p.c.) was on Oct 31, 2001.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 20, 60, 180 mg/kg bw/d
Basis:
nominal conc.
No. of animals per sex per dose:
25 time-mated female rats per dose, age ca. 70 d at beginning of the study (day 0, detection of sperm), were used.
Body weights ranged between 143 to 186 g on day 0 post coitum (p.c.).
Control animals:
yes, concurrent vehicle
Details on study design:
Only pregnant dams were used for the calculations of mean maternal food consumption, body weight and body weight change.
Only pregnant dams with scheduled sacrifice (day 20 p .c.) were taken for the calculation of mean gravid uterine weights,
mean net maternal body weight change (corrected body weight gain) and summary of reproduction data.

In this study the following females were partially or totally excluded from the above mentioned calculations:
Test group 0 (0 mg/kg bw/d): females Nos. 1, 9 and 15 - not pregnant
Test group 1(20 mg/kg bw/d): female No . 33 - not pregnant
Test group 2 (60 mg/kg bw/d): females Nos . 57 and 62 - not pregnant
Test group 3 (180 mg/kg bw/d): female No. 79 - not pregnant

Thus, according to the requirements of the most relevant OECD test guidelines which are listed in chapter 2 .3., each test group including
the controls contained a sufficient number of females with implantation sites at necropsy (" . . . .approximately 20, but not fewer than 16 females with implantation sites").

Examinations

Maternal examinations:
During the conduct of the study the animals were examined at least daily for clinical symptoms. Mortality was checked twice a day or once
on Saturday/ Sunday. Food and water consumption, body weight on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c..
Corrected body weight gain was calculated after terminal sacrifice as [bw d 20- (uterus weight + bw d 6 p.c.)].
Dams were sacrificed on day 20 p.c.. Necropsy included gross pathology assessment weight determinations of the unopened
uterus and the placentae.
Ovaries and uterine content:
Uterus and ovaries were removed with subsequent examination and recording of:
unopened uterus weight; no. of corpora lutea; no. and classification of implantation sites as live fetuses or dead implantations
(early, late resorptions, dead fetuses). Calculation of conception rate and pre- and postimplantation losses.

Fetal examinations:
Fetuses were weighed and sexed by anogenital distance observation (which was later confirmed by internal examination of those fetuses
which were preserved in BOUIN's solution), and macroscopically examined (viability, condition of placenta, fetal membranes, umbilical cords,placental weight). Approx. 50% of all fetuses were subjected to soft tissue examinations after fixation in BOUIN's solution, the other
50% of fetuses was examined for skeletal changes.
Statistics:
Statistical evaluation of data included FISHER's Exact Test for conception rate, mortality of the dams, and number of litters with fetal findings; WILCOXON Test for proportions of fetuses with malformations and/or variations in each litter; and DUNNETT's Test for all other data
including water consumption and body weight gain.
Indices:
Calculations of conception rate and pre- and postimplantation losses were carried out:

- The conception rate ( in %) was calculated according to the following formula :
(number of pregnant animals/number of fertilized animals)x100

- The preimplantation loss (in %) was calculated according to the following formula :
(number of corpora lutea - number of implantations/number of corpora lutea)x100

- The postimplantation loss (in %) was calculated from the following formula :
(number of implantations - number of live fetuses/ number of implantations)x100
Historical control data:
Detailed historical control data were included in the study report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Only pregnant dams or pregnant dams sacrificed at schedule were used. Animals which were totally or partially excluded were: 3 non-pregnant control animals; one non-pregnant animal from each dose group. No mortality was seen in any of the groups. The group mean reproductive and fetal data can be found in table 1 below.

Low dose, 20 mg/kg bw/d: No substance related effects seen.
Intermediate dose, 60 mg/kg bw/d: No substance related effects seen, except slight but significant decrease of body weight gain on d 6-8 p.c.
High dose, 180 mg/kg bw/d:
- transient salivation in 6 females between days 15 - 19 p.c. and vaginal hemorrhage in one dam on day 20 p.c.
- statistically significantly decreased food consumption on days 6 - 8 p.c. (about 13% less food intake as in the concurrent control group)
- statistically significantly impaired body weight gain on days 6 - 8 p.c. (about 45% below controls) and on days 17 - 20 p.c. (33% - 34% below controls)
- statistically significantly lowered mean uterus weight (about 26% below controls)

Effect levels (maternal animals)

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

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No substance related effects were observed at 20 and 60 mg/kg bw/d. Sex distribution was comparable to control animals in all treatment
groups.

Embryotoxic and teratogenic effects were present at the high dose level of 180 mg/kg bw/d (see also table 2 below):
- statistically significantly increased number of resorptions (especially late ones) and consequent a statistically-significantly increased
postimplantation loss value (43 .4% versus 7.9% in the controls); 3 of the 24 pregnant dams resorbed all implants and had no live fetuses
at necropsy
- statistically significantly increased mean placental weights (about 22% above controls)
- markedly reduced mean fetal body weights (about 14% below controls) due to a higher number of stunted fetuses (so-called runts)
- statistically significantly increased rate of external malformations (anasarca and/or cleft palate); these findings occurred in 13 of 132
fetuses [= 9.8%] in 7 out of 21 litters [= 33%], i.e. 9.0% versus 0.0% affected fetuses/ litter in the control group
- statistically significantly increased rate of soft tissue variations (dilated renal pelvis, dilated ureter) with a total of 27.1 % versus 6.4%
affected fetuses/litter in the control group
- statistically significantly increased rate of skeletal malformations (e.g. shortened scapula, bent radius/ulna, malpositioned and bipartite
sternebra); these findings occurred in 7 of 73 fetuses [= 9.6%] in 5 out of 21 litters [= 24%], i.e. 7.8% versus 1.1% affected fetuses/litter in
the control group
- statistically significantly increased occurrence of several skeletal variations (mainly delays in the ossification process) with a total of 98.4%
versus 91.1 % affected fetuses/litter in the control group
- statistically significantly increased rates of total malformations (10.8% versus 0.6% affected fetuses/ litter in the control group), variations
(7 0.4% versus 52.0% affected fetuses/ litter in the control group) and unclassified skeletal cartilage observations (69.8% versus 50.8%
affected fetuses/ litter in the control group)


Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Clinical data:

Transient salivation was noted in 6/25 animals during days 15-19 p.c. starting few minutes after dosing and lasting 15 to 20 min. Vaginal hemorrhage on d 20 was noted in one animal. Salivation was regarded to be treatment related due to bad taste or local affection of the upper digestive tract, but was not assessed as an adverse or toxic effect.

Food intake, body weights:

Food intake was significantly reduced (-13% compared to controls) during a period at the beginning of treatment on d 6-8 p.c. Body weight gain was also statistically reduced compared to controls on d 6-8 p.c. (-45%) and d 17-20 p.c. (-34%). However, at the end of the treatment period body weight was comparable in all groups. Findings were regarded as being substance-induced and reflecting direct adverse effects on dams (d 6-8) and on fetuses (d 17-20; resorptions, lowered fetal body weight). Corrected body weight gain was comparable in all dose groups.

Table 1 Group mean reproductive and fetal data

 

Dose levels
(mg/kg bw/d)

0

20

60

180

Pregnancies on day 20

22

24

23

24

Conception rate

88

96

92

96

Dams with viable fetuses

22

22

23

21

Gravid uterus weight (g)

51.3 (9.52)

45.6 (16.65)

50.0 (11.57)

38.2 (16.81)**

Implantations/dam

9.7 (1.67)

9.0 (2.1)

9.7 (1.69)

9.3 (1.88)

Pre-implantation loss (%)

10.7 (14.07)

18.5 (20.84)

14.5 (13.33)

15.7 (12.46)

Post-implantation loss (%)

7.9 (10.13)

14.8 (28.31)

9.6 (11.77)

43.4 (34.09) **

Resorptions (total)

0.8 (1.05)

0.9 (1.23)

0.9 (1.12)

3.8 (3.06) **

Live fetuses/dam

8.9 (1.61)

8.8 (1.68)

8.8 (1.93)

6.3 (3.15) **

Fetal weight (g)
(all viable)

3.7 (0.27)

3.6 (0.2)

3.6 (0.32)

3.2 (0.27) **

Placental weights (mg) (all viable fetuses)

460 (62)

450 (46)

490 (57)

560 (173) **

Sex ratio (% males)

53

41

52

46

SD in brackets

** p < = 0.01 (Dunnett test) (two-sided)

 

Table 2 Summary of all classified fetal external, soft tissue, and skeletal observations

 

Parameter

 No. and (%) fetuses at (mg/kg bw/d)

0

20

60

180

No. litters evaluated

22

22

23

21

No. fetuses evaluated

195

194

202

132

Total malformations, mean (%) (affected fetuses/litter)

0.6 (3.05)

1.1 (3.68)

0.5 (2.32)

10.8 (14.67) **

Total variations, mean (%)
(affected fetuses/litter)

52 (11.51)

50 (13.24)

61 (15.8)

70.4 (20.64) **

Unclassified skeletal cartilage observations, mean %, (affected fetuses/litter)


50.8 (29.2)


42.9 (38.11)


51.2 (28.95)


69.8 (28.79) **

External malformations, mean
- litter incidence (%)
- affected fetuses/litter (%)


0
0


0
0


0
0


33 ##
9 (15.08) **

Skeletal malformations, mean
- litter incidence (%)
- affected fetuses/litter (%)


4.5
1.1 (5.33)


9.1
2.3 (7.36)


4.3
0.9 (4.17)


24
7.8 (15.95) *

Soft tissue variations, mean
- affected fetuses/litter (%)

6.4 (16.25)

9.2 (17.02)

22.7 (29.69) *

27.1 (35.05) *

Skeletal variations, mean
- affected fetuses/litter (%)

91.1 (14.91)

87.2 (16.1)

94.2 (9)

98.4 (7.27) *

SD in brackets
* p < = 0.05 (Wilcoxon-test, one-sided), ** p < = 0.01 (Wilcoxon-test, one-sided)
## p < = 0.01 (Fischer’s exact test, one-sided)

 

Applicant's summary and conclusion

Executive summary:

The developmental toxicity of imidazole was studied in accordance to OECD TG 414 in rats at dose levels of 20, 60 and 180 mg/kg bw/d.

Maternal toxicity was noted exclusively at 180 mg/kg bw/d as substantiated by significantly reduced food intake at initiation of treatment and impaired body weight gains on days 6-8 p.c.. No signs of maternal toxicity were seen at 60 mg/kg bw/d and below. Fetal development was adversely affected at 180 mg/kg bw/d as substantiated by the high rate of late resorptions (3/24 animals showing complete resorption) which resulted in an elevated postimplantation loss and reduced fetal body weights.

At 180 mg/kg bw/d selective teratogenicity was indicated by increased occurrence of external and skeletal malformations and variations of which anasarca, cleft palate, shortened scapula, and incomplete or delayed ossifications were the most prominent. No increases were noted at 20 or 60 mg/kg bw/d.

Based on these findings NOAEL for maternal toxicity and for prenatal developmental toxicity is 60 mg/kg/d.

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