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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
equivalent or similar to
Guideline:
other: Commission Regulation (EC) No 440/2008, Part B: Methods for the determination of toxicity and other health effects: Two-Generation Reproduction Toxicity Study
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Groups of 25 pregnant females received the test substance by oral gavage (control and 3 test groups) from gestation day 6 through day 3 of lactation. The dams and pups were sacrificed on post-natal day 4. Examinations in adult females included clinical signs, parturition and lactation behaviour, body weight, food consumption and gross pathology. Pup examinations included pup status, litter size and external examination at birth, viability, clinical signs, body weight, necropsy and microscopic examination of the great blood vessels of the heart.
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-Methylimidazol
- Test substance number: 02/0098-2
- Description: solid / colorless to yellow
- Batch: 05649175L0
- Purity: 99.8 corr. %
- Storage: room temperature
- Homogeneity: Given
- Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor
- Expiry date: 19 September 2012

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Female rats, time-mated by the breeder (supplied at noon on the day of evidence of mating)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH.
- Age at arrival: 10-12 weeks.
- Body weight at arrival (gestation day 0): 142.2 – 183.1 g
- Housing: In Makrolon type M III cages, 1 animal per cage (exception: dams were housed together with their litter), with dust-free wooden bedding. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. Wooden gnawing blocks (Type NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, were added for environmental enrichment.
- Diet: Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, available ad libitum.
- Water: drinking water (from water bottles), available ad libitum.
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% carboxymethylcellulose in drinking water
Details on exposure:
- Amount of vehicle: 10 mL/kg body weight.
- Preparation and storage of test substance formulations: The test substance was weighed in a calibrated beaker, topped up with the vehicle and intensely mixed with a homogenizer. During administration, the formulations were kept homogeneous with a magnetic stirrrer. Fresh formulations were prepared at intervals which guarantee that the test substance in the vehicle remains stable. The formulations were stored in a refrigerator.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses for stability, homogeneity and concentration control of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany, in compliance with the Principles of Good Laboratory Practice. The samples were analysed using a validated HPLC method.
The stability of the test substance in the vehicle upon storage in a refrigerator for a period of 7 days was demonstrated before the start of the toxicity study in a similar batch (BASF Project No. 01Y0098/02Y003). All determined concentrations in the stability samples were in the range between 90% and 110% of the nominal concentrations.
Samples of the test substance preparations, taken at the beginning of the administration period, were sent to the analytical laboratory once during the study period for verification of the concentration and homogeneity: one sample of the control and low-dose formulations, and three samples (withdrawn from the top, middle and bottom of the preparation container) of the low- and high-dose formulations, taken from the container with a magnetic stirrer running. The measured concentrations were in the range between 90% and 110% of the nominal concentration for the mid-dose sample and the high-dose samples. The determined mean values for the low-dose samples were in the range from 116.6 % - 122.4 % (repeat and initial analysis, respectively), which was slightly above the specification limit of 110 %. The low relative standard deviations (maximally 2.4%) indicated that the test substance was homogeneously distributed in the vehicle.
Details on mating procedure:
Time-mated females were obtained from the animal supplier. The animals were supplied at noon on the day of evidence of mating; this day is referred to as GD 0 and the following day as GD 1.
Duration of treatment / exposure:
From implantation to one day prior to sacrifice (gestation day 6 through post-natal day 3).
Frequency of treatment:
Once daily at about the same time in the morning (females in labor were not treated).
Doses / concentrations
Remarks:
Doses / Concentrations:
2, 10 and 50 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
25 females per dose. This group size (22-25 pregnant females) was selected to attain statistical power equivalent to that of a full-scale generation study (e.g. OECD 416).
Control animals:
yes, concurrent vehicle
Details on study design:
- Selection of doses: Based on the results of a previously conducted reproduction/developmental toxicity screening test (OECD421) in which adverse developmental effects (dissecting aneurysms in the great vessels of the heart) were observed up to the lowest dose tested (50 mg/kg bw/day).
- Parturition: The females were allowed to deliver and rear their pups until day 4 after parturition.
- Termination in-life phase: Dams and their pups were sacrificed on PND 4 and subjected to post mortem examinations.

Examinations

Maternal examinations:
- Mortality: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
- Clinical signs: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hours period from about 15:00 h of one day until about 15:00 h of the following day.
- Food consumption: Food consumption during pregnancy was determined for GD 0-6, 6-8, 8-10, 10-13, 13-15, 15-17 and 17-20. Food consumption of the females which gave birth to a litter was determined for PND 1-4. Food consumption was not determined in the females without litter during the lactation period.
- Body weight: Body weights during pregnancy were determined on GD 0, 6, 8, 10, 13, 15, 17 and 20. Body weights of the females which gave birth to a litter were determined for PND 0 and PND 4. Body weights were not determined in the females without litter during the lactation period.
- Post mortem examinations: On the day after the last test substance administration the animals were anesthetized with isoflurane (after overnight fasting), sacrificed by cervical dislocation and assessed by gross pathology. Animals without litter were anesthetized with isoflurane, sacrificed by cervical dislocation and assessed by gross pathology shortly after the expected day of delivery. The apparently non-pregnant uteri were stained for about 5 minutes in 1% ammonium sulfide solution according to the method of SALEWSKI (Salewski, E.; 1964). Then the uteri were rinsed carefully with 0.9% NaCl solution.
- Litter observations: see under 'Fetal examinations'.
Fetal examinations:
Litter observations:
- Pup number and status at delivery: All pups delivered were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also examined for macroscopically evident changes. Pups that died before this initial examination were defined as stillborn pups.
- Pup viability/mortality: In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index PND 0-4 was calculated.
- Sex and sex ratio: On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 4 after birth.
- Clinical observations: All live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.
- Body weight: The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
- Post mortem examinations of pups sacrificed on schedule: On PND 4, the pups were sacrificed under isoflurane anesthesia with CO2. The pups were examined externally and their organs were assessed macroscopically, paying particular attention to the heart and great vessels of the heart, without removing the organs (except the thymus).
- Post mortem examinations of prematurely died or sacrificed pups: Pups that died or were sacrificed in a moribund state were eviscerated and examined for possible defects and/or the cause of death using appropriate methods, paying particular attention to the heart and great vessels of the heart, without removing the organs (except the thymus).
- Preservation: All pups were preserved in toto (except the thymus) in 4% neutral buffered formaldehyde solution and after 24 - 48 hours they were transferred to 70% alcohol solution for further preservation.
- Histopathology: After fixation, the great blood vessels of the heart were dissected in an appropriate way and processed histotechnically in two paraplast blocks: Block 1 included the base of the heart, aortic arch (aorta ascendens and aorta descendens), pulmonary trunk, and ductus arteriosus. Block 2 included three sections of the abdominal aorta (at the diaphragmatic insertion, region of kidneys [A. renalis] and region of the caudal bifurcatio aortae). Histotechnical processing was followed by light microscopical examination of the aortic arch (block 1; Hart stain combined with Masson-Goldner-Trichrome stain for detection of elastic and collagen fibers of the vessel walls) of all pups/test group, the abdominal aorta (block 2; paraplast embedding) of all pups/test group, and gross lesions (only macroscopic aneurysms/dilations) in all affected pups/test group. Immunohistochemistry investigations (antibody against collagen type I and III) were conducted on the aortic arch of selected control and high-dose pups only. The control pups were selected randomly (4 male and 6 female pups). In the high-dose group, pups with macroscopic findings suspected of aneurysms (2 male and 3 female pups) and additional randomly selected pups wiithout macroscopic or microscopic findings (2 male and 3 female pups) were used. Pups that died or were sacrificed in a moribund state were processed histotechnically and assessed like control pups.
Statistics:
The following statistical methods were used to analyze the data:
- Food consumption, body weight and body weight change (parental females and pups; litter means were used for pup weights), duration of gestation, number of delivered pups per litter, live pups on day x: Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means
- Females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups,: Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions
- Viability Index: Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians
Indices:
- Female fertility index (%) = (number of females pregnant* / number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

- Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant*) x 100
* defined as the number of females with implants in utero

- Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

- Viability index (%) = (number of live pups on day 4 after birth / number of live pups on the day of birth) x 100

- Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
- Mortality: There were no test substance-related mortalities in any of the parental females in any of the groups.
- Clinical observations: No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the parental females during the whole study. One sperm positive low-dose female (2 mg/kg bw/d) and two control females (Nos. 4 and 21) did not deliver F1 pups.
- Food consumption: Food consumption of the parental females in all test substance-treated groups (test groups 1 - 3; 2, 10 and 50 mg/kg bw/d) was comparable to the concurrent control group during the entire study period. Food consumption was statistically significantly decreased on several occasions during pregnancy in test groups 1 and 2 (2 and 10 mg/kg bw/d). These minor and non-dose related changes were regarded as spontaneous in nature.
- Body weight data: Mean body weights and mean body weight change of the parental females in all test substance-treated groups (test groups 1 - 3) were comparable to the concurrent control group during the entire study period.
- Female reproduction and delivery data: All sperm positive rats delivered pups or had implants in utero with the following exceptions: control female No. 4, control female No. 21, low-dose female No. 33, which were not pregnant, and therefore, did not deliver F1 pups.
The mean duration of gestation was similar in all test groups (i.e. between 22.1 and 22.2 days). The gestation index was 100% in all test groups. The mean number of F1 pups delivered per dam remained unaffected (9.7 / 8.8 / 9.8 and 9.3 pups/dam at 0, 2, 10 and 50 mg/kg bw/d). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% in control and test groups 1 and 3, as well as 99.6% in test group 2. Moreover, the number of stillborn pups was comparable between the groups.
- Maternal necropsy observations: One parental animal showed a spontaneous finding at gross necropsy (dilated renal pelvis). This finding occurred without any relation to dosing.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
- Pup number and status at delivery: The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
- Pup viability/mortality: The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 100% (control and test groups 2 and 3) and 99.6% (test group 2) without showing any association to the treatment.
- Sex ratio: The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
- Pup clinical observations: There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups. One male pup (dam No. 31, pup No. 1) of dose group 1 (2 mg/kg bw/d) showed a spontaneous finding (short tail).
- Pup body weight data: Mean body weights and mean body weight change of the male and female pups in all test substance-treated groups (test groups 1 - 3) were comparable to the concurrent control group during the entire study period. One male runt was seen in the control, one male and one female runt were seen in test group 1, one male runt was seen in test group 2, two male and four female runts were seen in test group 3.
- Pup necropsy observations: The macroscopic examination of pups at necropsy revealed a number of findings concerning the pericardial vessels in the all-dose groups. More than one of macroscopic findings in the great vessels at the base of the heart (aorta, ductus arteriosus, pulmonary trunk and carotid artery) were simultaneously observed in individual pups. A table showing the numbers of pups with macroscopic findings in the great vessels at the base of the heart is given under 'Any other information on results incl. tables'. A few other pups showed spontaneous findings at gross necropsy, such as dilated ureter, red discolored testis, dilated renal pelvis and white discolored liver lobe. These spontaneous findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences.The two decedent pups (No. 54-11 and 74-5) of test group 2 (10 mg/kg bw/d) showed no abnormalities.

- Histopathology: Dissecting aneurysms result from rupture of the tunica intima occurring in vivo allowing the entry of large amounts of blood through the tunica media that accumulate over time and make the adventitia protrude outside the arterial wall. The criteria for differentiating dissecting aneurysms (intramural blood extravasation occurring in vivo) from artifacts (postmortem blood extravasation occurring during necropsy due to tearing of blood vessels) were taken from Treumann et al., 2011 (Silke Treumann, Steffen Schneider, Sibylle Gröters, Nigel P. Moore and Paul J Boor (2011). Spontaneous occurrence of dissecting aneurysms in the region of the ductus arteriosus in four-day-old Wistar rat pups. Toxicologic Pathology 39: 969-974 ).
In the present study, dissecting aneurysms were characterized by a focal bulging of the arterial wall due to accumulation of densely packed erythrocytes between the outer tunica media and the adventitia (dissecting effect) and additional delicate residues of torn elastic fibers. Hemorrhages were characterized by discrete focal accumulation of erythrocytes within the tunica media that did not cause either bulging of the arterial wall or tearing of elastic fibers. Large aneurysms in some pups extended along different vessels connected with each other (e.g. along ductus arteriosus connecting the pulmonary trunk with the descendent portion of the aorta). Most of the aneurysms occurred in the region of the ductus arteriosus connecting with the descending aorta. Most of the hemorrhages were seen in the descending aorta. Some pups had more than one aneurysm and others had more than one hemorrhage. However, the two findings aneurysm and intramural hemorrhages did never occur simultaneously in the same pup, which means that either aneurysm(s) or intramural hemorrhage(s) occurred in the individual animal. Only single pups in each litter were affected, with one exception each in test group 2 (10 mg/kg bw/d) and test group 3 (50 mg/kg bw/d). In test group 2, two females (Nos. 66-6 and 66-7) of the same litter and in test group 3 (50 mg/kg bw/d), two males (Nos. 91-3 and 91-5) of the same litter were affected. In each of these litters one pup had an aneurysm and the other pup had a hemorrhage. In male pups, histopathology revealed that all aneurysms correlated with the finding “dilation” of the vessels observed at gross pathology. Conversely, in one female pup of test group 2 (10 mg/kg bw/d) and two female pups of test group 3 (50 mg/kg bw/d), macroscopic findings in the vessels had no histopathological correlate and thus the macroscopic findings were regarded as artifacts. The immunohistochemical stain with antibodies against collagen type I and III did not reveal differences between the collagen fibers of control and treated animals with or without dissecting aneurysms. A table showing the numbers of aneurysms and hemorrhages for the various locations is given under 'Any other information on results incl. tables'.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Pup macroscopic findings in the great vessels at the base of the heart

 

Number of findings

Male pups (F1)

Female pups (F1)

Test group

(mg/kg bw/d)

0

(0)

1

(2)

2

(20)

3

(50)

0

(0)

1

(2)

2

(10)

3

(50)

No. of examined pups

99

112

123

114

123

99

119

118

No. of pups with macroscopic findings in the great vessels at the base of the heart

0

1

2

2

0

0

2

3

Aorta

 

 

 

 

 

 

 

 

  - Dilation

 

1

1

1

 

 

2

1

  - Discoloration

 

 

 

 

 

 

 

1

Ductus arteriosus

 

 

 

 

 

 

 

 

  - Dilation

 

1

2

2

 

 

 

1

Pulmonary trunk

 

 

 

 

 

 

 

 

  - Dilation

 

 

1

1

 

 

 

1

Carotid artery

 

 

 

 

 

 

 

 

  - Dilation (right)

 

 

1

 

 

 

 

 

  - Narrow (left)

 

 

1

 

 

 

 

 

 

  

Pup microscopic findings: number of aneurysms and hemorrhages for the various locations

 

 

Male pups (F1)

Female pups (F1)

Test group

(mg/kg bw/d)

0

(0)

1

(2)

2

(10)

3

(50)

0

(0)

1

(2)

2

(10)

3

(50)

No. of examined pups

99

112

123

114

123

99

119

118

Aorta

 

 

 

 

 

 

 

 

  - Aneurysm

 

1

1

1

 

 

1

1

  - Hemorrhage

 

 

 

2

2

3

1

 

Ductus arteriosus

 

 

 

 

 

 

 

 

  - Aneurysm

 

1

2

2

 

 

 

1

  - Hemorrhage

 

 

 

 

2

 

 

 

Pulmonary trunk

 

 

 

 

 

 

 

 

   - Aneurysm

 

 

1

1

 

 

 

 

Carotid artery, right

 

 

 

 

 

 

 

 

  - Aneurysm

 

 

1

 

 

 

 

 

 

Historical control data:

An evaluation of 1016 untreated Wistar rat pups culled at PND 4, from reproductive toxicity studies conducted in the laboratory, revealed low incidences of aneurysms and hemorrhagic lesions, affecting 0.2% of all the offspring that were evaluated (Treumann et al., 2011). Since the analysis was based on pups culled from other studies, the litter-based incidence of these lesions in unexposed animals cannot precisely be determined. Nevertheless, the findings indicate that, although rare, dissecting aneurysms may occur as spontaneous lesions, such that the low incidence in group 1 (1 pup or 0.5% of total offspring affected) cannot be conclusively attributed to treatment.

Reference: Silke Treumann, Steffen Schneider, Sibylle Gröters, Nigel P. Moore and Paul J Boor (2011) Spontaneous occurrence of dissecting aneurysms in the region of the ductus arteriosus in four-day-old Wistar rat pups. Toxicologic Pathology 39: 969-974.

Applicant's summary and conclusion