Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test, July 2000)
GLP compliance:
yes
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 area-% (report No: 11L00194)
- Storage: room temperature
- Homogeneity: Given (vis.)
- 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
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at start of treatment: 11-13 weeks
- Weight at start of treatment: Males: 297.8-324.2g, Females: 195.0-238.7g
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²). During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. Pregnant animals and their litters were housed together until PND 4. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). 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 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 the start of the administration period and therafter at maximum intervals of 7 days, which took into account the analytical results of the stability verification.
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00-09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".
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).
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. Three samples (one from the top, middle and bottom) were taken from the beaker with a magnetic stirrer running. The measured concentrations were in the range between 90% and 110% of the nominal concentration for all samples. The low relative standard deviations (4.5% and 1.4% for the low- and high-concentration, respectively) indicated that the test substance was homogeneously distributed in the vehicle.

Duration of treatment / exposure:
Males were exposed for 28 days, namely during 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females were exposed during 2 weeks prior to mating, during mating (max. of 2 weeks), during gestation and during 4 days of lactation (last dose on the day prior to scheduled necropsy).
Frequency of treatment:
Once daily at about the same time in the morning (females in labor were not treated).
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 150 and 500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Selection of doses: By request of the sponsor.
- Parturition: The females were allowed to litter and rear their pups until day 4 after parturition. Pups were sacrificed on PND 4 and gross necropsied.

Examinations

Parental animals: Observations and examinations:
- Mortality / Viability: 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 Saturday, Sunday and 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: Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
Food consumption was not determined after the 2nd premating week (F0 animals) during the mating period (male and female F0 animals); Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14, 14-20; Food consumption of F0 females which gave birth to a litter was determined for PND 1-4. Food consumption was not determined in females without positive evidence of sperm during mating and gestation periods and in females without litter during the lactation period.
- Body weight data: Body weight was determined three days before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20; Females with litter were weighed on the day of parturition (PND 0) and on PND 4; Females without a litter and females without positive evidence of sperm in the vaginal smear were weighed weekly (these body weight data were solely used for the calculations of the dose volume).
- General reproduction and delivery data: The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs. Male and female mating and fertility indices, gestation index, gestation length and post-implantation loss were calculated. The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters.
Litter observations:
- Pup number and status at delivery: All pups delivered from the F0 parents (F1 litter) 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: The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.
- Body weights: 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.
Postmortem examinations (parental animals):
- Necropsy: All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs. Two females (No. 136 and 140 of the high-dose group) which died intercurrrently were necropsied as soon as possible after their death and assessed by gross pathology.
- Terminal body weight and organ weights: The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Epididymides, Testes.
- Tissue preservation: The following organs or tissues were fixed in 4% buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Cervix, Coagulating glands, Epididymides (modified Davidson’s solution), Ovaries (modified Davidson’s solution), Oviducts, Prostate gland, Seminal vesicles, Testes (modified Davidson’s solution), Vagina, Uterus. The ovaries of the females that died intercurrently were fixed in 4% buffered formaldehyde solution.
- Histopathology: Fixation was followed by histotechnical processing (HE staining) and examination by light microscopy of the Testes, Epididymides and Ovaries of all animals of the control group and the high-dose group.
Postmortem examinations (offspring):
- Necropsy: All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically, paying particular attention to the heart and aortic vessels. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and examined for possible defects and/or the cause of death, paying particular attention to the heart and aortic vessels. Moribund pups that were sacrificed before PND 4 were also eviscerated and assessed macroscopically, with special attention given to the basis of the heart and the great vessels.
- Preservation: All pups with findings and 10 control pups (5/sex) were preserved in toto in 4% neutral buffered formaldehyde after gross examination for further histopathological examination. All remaining pups without notable findings or abnormalities were discarded after their macroscopic evaluation.
- Histopathology: Fixation was followed by histotechnical processing and examination by light microscopy of the basis of the heart and great vessels of 5 male and 5 female pups of the control group and all affected pups of the low-, mid- and high-dose groups. The pups were stained with Hart stain combined with Masson-Goldner-Trichrome stain for detection of elastic and collagen fibers of the vessel walls.
Statistics:
The following statistical methods were used to analyze the data:
- Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and percentage postimplantation loss, number of pups delivered per litter: Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means.
- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups canniballized, pups sarificed moribund, viability index, number of litters with affected pups at necropsy: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions.
- Proportions of affected pups per litter with necropsy observations: Pairwise comparison of each dose group with the control group using WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Anesthetized animals and organ weights: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Reproductive indices:
For each group, the following calculations were performed:
- Male mating index (%) = (Number of males with confirmed mating/Number of males placed with females) x 100
- Male fertility index (%) = (Number of males proving their fertility/Number of males placed with females) x 100
- Female mating index (%) = (Number of females mated / Number of females placed with males) x 100
- Female fertility index (%) = (Number of pregnant females / Number of females mated) x 100
- Gestation index (%) = (Number of females with live pups on the day of birth / Number of pregnant females) x 100
- Post-implantation loss (%) =( (Number of implantations - number of pups delivered) / number of implantations) x 100
Offspring viability indices:
For each group, the following calculations were performed:
- 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 post partum / Number of pups born alive) x 100
- Sex ratio day 0/4 = ( 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: P0 (first parental generation)

Details on results (P0)

MORTALITY
Two female animals of test group 3 (500 mg/kg bw/d) were found dead during the lactation period. High-dose female No. 136 was found dead on PND 2. For this animal some preceding abnormal clinical findings were recorded, such as yellowish discolored urine, light brown discolored feces, insufficient maternal care of pups and not consumed placentas. High-dose female No. 140 was found dead on PND 3 after showing chromodacryorrhea, piloerection, yellowish discolored urine, light brown discolored feces and a poor general state. Furthermore, this animal had undelivered pup(s) palpable in its abdomen on GD 22 and on PND 0, it showed salivation after treatment, its pups were not properly nursed and the umbilical cords were not cut. As a consequence, no more pups were alive on PND 1. There were no pathological findings which could explain these premature deaths.
There were no test substance-related or spontaneous mortalities in the F0 parental animals of test groups 1 and 2 (50 and 150 mg/kg bw/d).
CLINICAL SIGNS
All 10 male and 10 female F0 parental animals of the high-dose group (500 mg/kg bw/d) showed transient salivation during major parts of the treatment period, inclusive gestation and lactation. In the mid-dose group (150 mg/kg bw/d) nearly all female animals (7 out of 10 at a maximum) showed salivation after treatment during the premating, gestation and lactation periods. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 10 minutes) and was initially observed during study week 1. Since this salivation was transient and occurred directly after dosing, it was probably due to the irritation and/or taste of substance administration, and therefore was not assessed as adverse.
All high dose male and female F0 parental animals (500 mg/kg bw/d) had yellowish discolored urine during the entire study period, covering premating, gestation and lactation periods. In the mid-dose group (150 mg/kg bw/d) for all male and female F0 parental animals the discolored urine was recorded from study week 2 onwards until scheduled sacrifice. Furthermore, out of 10, respectively, light brown discolored feces were noticed in 3 high-dose females during GD 21-22 and 8 high-dose females during PND 0-4. This discoloration of urine and feces was probably a result of the metabolism and subsequent excretion of the test compound. While this effect was substance-dependent, it was judged not to be adverse.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals at 50 mg/kg bw/d during the different study periods.
BODY WEIGHT
Mean body weights and mean body weight gain of the F0 males in test groups 1-3 (50, 150 or 500 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study.
On the whole, mean body weights and mean body weight gain of the high-dose F0 females (500 mg/kg bw/d) were comparable to the control throughout the entire premating, gestation and lactation periods. However, if calculated for GD 0-20, mean body weight gain was statistically significantly reduced in this test group (-18%) and, on PND 0, mean body weights were slightly, but statistically significantly, decreased in these animals (-7%).
Mean body weight and mean body weight gain of the F0 females in test groups 1 and 2 (50 and 150 mg/kg bw/d) were comparable to the concurrent control throughout the entire premating, gestation and lactation periods.
FOOD CONSUMPTION
Food consumption of the substance-treated F0 parental males (test groups 1-3; 50, 150 or 500 mg/kg bw/d) was comparable to the concurrent control group throughout the entire study period.
Food consumption of the high-dose F0 parental females (500 mg/kg bw/d) was slightly reduced during premating, gestation and lactation periods, attaining statistical significance in premating week 0-1 (13% less in comparison to the control) and PND 1-4 (20% less).
Low- and mid-dose F0 parental females (50 and 150 mg/kg bw/d) did not show any test substance-related changes in food consumption during the whole treatment period.
REPRODUCTIVE PERFORMANCE
- Copulation was confirmed for all F0 parental males and females which were placed with females to generate F1 pups. Thus, the male and female mating index was 100% in all groups including the controls. The mean duration until sperm was detected (GD 0) varied between 2.3 and 3.3 days without any relation to dosing.
- Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. Two control males (Nos. 3 and 9) did not generate F1 pups. These two apparently infertile male rats did not show relevant gross lesions, which could explain the infertility.
One female rat (No. 111 - 50 mg/kg bw/d) was not detected as sperm positive, but had pups. All sperm positive rats delivered pups or had implants in utero except for two control females (Nos. 103 and 109, mated with males Nos. 3 and 9). The two non-pregnant control females had no relevant gross lesions. The male and female fertility index varied between 80% (control) and 100% (test groups 1-3). These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
- The gestation index was 100% in all test groups. Although the high-dose value (22.5 days) was slightly, but statistically significantly increased, the mean duration of gestation was comparable between the test substance-treated groups and the control group (i.e. between 21.9 and 22.5 days). The longer gestation time in the high-dose group was similar, when compared to the historical control data for this rat strain from OECD screening studies (21.6 - 22.4 days) and still within the historical control range collected from multi-generation studies with this rat strain (21.5 - 22.5 days). However, considering the two high-dose dams which died having parturition difficulties, and influence of the test substance on normal term delivery cannot be excluded.
- Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (12.9 / 12.8 / 13.0 / 11.8 implants/dam in test groups 0-3 (0, 50, 150 and 500 mg/kg bw/d)). There were no statistically significant differences in post-implantation loss between the groups (8.2% / 7.5% / 12.0% / 9.2%), and the mean number of F1 pups delivered per dam remained unaffected (11.8 / 11.9 / 11.5 and 11.1 pups/dam at 0, 50, 150 and 500 mg/kg bw/d).
- Delivery data are presented under ‘Details on results (offspring)’.
ORGAN WEIGHTS
The mean absolute and relative organ weights (testes and epididymides) did not show significant differences when compared to the control group 0.
GROSS PATHOLOGY AND HISTOPATHOLOGY
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: F1 generation

Details on results (F1)

PUP NUMBER AND STATUS AT DELIVERY
The rate of liveborn pups was statistically significantly reduced in the high-dose group (500 mg/kg bw/d), as indicated by a reduced live birth index (100% in test groups 0 and 1, 97% in test group 2 and 90%** [p<=0.01] in test group 3) in this group. Moreover, the number of stillborn pups was statistically significantly increased in the high-dose group (0 / 0 / 4 / 11** [p<=0.01] pups/dam at test groups 0-3). This fact is mainly caused by high-dose dam No. 140, which had 7 stillborn pups (3 male / 4 female) in its litter (12 pups in total). However, the respective values were clearly outside the historical control range (HCD range: 0.0 - 7.3 stillborn pups/dam; 93 - 100% live birth index) and thus reflects a treatment-related effect in this dose group. The mean number of delivered pups per dam in the high-dose group (11.1) did not differ significantly from that in the control group (11.8).
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were comparable between test groups 0, 1 and 2.
VIABILITY/MORTALITY
Statistically significantly more pups died in the high-dose litters than control. That is, 28 died in the high-dose group (500 mg/kg bw/d), compared to none dead in the control group. Furthermore, 3 pups were cannibalized in the high dose group in comparison to one cannibalized pup in the control and test group 1, respectively, and 2 cannibalized pups in test group 2. Consequently, the viability index as indicator for pup mortality during lactation (PND 0-4) was significantly lower in test group 3 (59%** [p<= 0.01]) than in the control and test groups 1-2 (i.e. 99% / 98% / 97% at 0, 50 and 150 mg/kg bw/d).
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.
CLINICAL SIGNS
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
BODY WEIGHT
Mean pup body weight and pup body weight change of the high-dose F1 pups were slightly reduced during lactation period, however, without attaining statistical significance. Mean pup body weight and pup body weight change of the low- and mid-dose F1 pups were statistically comparable to the concurrent control group.
There were no runts in the control group. Respectively, two female runts were seen in each of test groups 1 and 2 (50 and 150 mg/kg bw/d), despite the lack of alterations in mean pup body weight parameters. Therefore these findings are considered spontaneous. However, the two male and four female runts seen in test group 3 (500 mg/kg bw/d) correspond to marginal reductions in pup body weight and pub body weight change, indicating a possible substance-related effect.
GROSS PATHOLOGY (OFFSPRING)
Aneurysms were present at different levels of the aorta (ascending, descending or aortic arch), in the region of ductus arteriosus and the pulmonary trunk. Frequently, aneurysms were observed simultaneously at different sites in the same pup. The number of affected pups increased from test group 1 (50 mg/kg bw/d) to test group 3 (500 mg/kg bw/d) in both male and female pups. Dilation of the aorta and heart was seen in 2 males and 2 females of test group 3 (500 mg/kg bw/d). A table showing the numbers of pups with macroscopic findings and aneurysms is given under ‘Any other information on results incl. tables’.
HISTOPATHOLOGY
Dissecting aneurysms were found in the aorta (ascending, descending and aortic arch), in the ductus arteriosus and the pulmonary trunk. Several animals showed simultaneously aneurysms at these different sites. In most cases, histopathology correlated with the aneurysms detected at gross pathology. The aneurysms were characterized by bulging of the affected vessel due to a cavity within the artery wall filled with blood and dissecting the elastic fibers of the medial layers. Mostly they were observed in the descending aorta. Occasionally, the aneurysms were compressing the lumen of the affected vessel. Dilation of the heart or of the aorta was noted only in single male and female pups of test group 3 (500 mg/kg bw/d). A table showing the numbers of examined control pups as well as treated pups with gross lesions (mostly aneurysms) is given under ‘Any other information on results incl. tables’.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Gross lesions in pups:

 

Test group

(mg/kg bw/d)

Male pups

Female pups

0

1

2

3

0

1

2

3

(0)

(50)

(150)

(500)

(0)

(50)

(150)

(500)

No. of pups with findings

0

1

8

20

0

1

7

24

No. of pups with aneurysms

0

1

8

18

0

1

6

24

Microscopic findings in pups:

Test group

(mg/kg bw/d)

Male pups

Female pups

0

1

2

3

0

1

2

3

(0)

(50)

(150)

(500)

(0)

(50)

(150)

(500)

No. of examined pups

5

1

8

20

5

1

7

24

No. of pups with aneurysm

0

1

8

16

0

1

6

21

       No of pups with one location

 

1

5

10

 

1

3

16

       No of pups with multiple locations

 

 

3

6

 

 

3

5

Aorta

 

1

7

11

 

1

6

19

       descending

 

1

7

10

 

1

6

19

       ascending

 

 

 

1

 

 

 

1

       arch

 

 

 

1

 

 

 

 

Ductus arteriosus

 

 

4

9

 

 

3

3

Pulmonary trunc

 

 

3

5

 

 

3

5

Applicant's summary and conclusion