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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
Please refer to read across justification (chapter 13)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
12 May 1981
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
structural analogue: see confidential details on test materials

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage MI
- Age at study initiation: 64 days
- Mean weight at study initiation and range: 222.9 g (195.1-255.4g)
- Fasting period before study:
- Housing: Individually, except during mating, in stainless steel suspended cages
- Diet (e.g. ad libitum): Certified Rodent Diet (mash), No. 5002; (PMI Feeds Inc., St. Louis, Missouri), ad libitum.
- Water (e.g. ad libitum): tap water, ad libitum.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24°C
- Humidity (%): 46-72%
- Photoperiod (hrs dark / hrs light): 12 h / 12 h


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Appropriate amounts of test material were mixed with the vehicle (corn oil) to provide concentration levels of 12, 36 and 108 mg/mL for the 60, 180, and 540 mg/kg/day groups, respectively.
- In addition, an appropriate amount of vehicle was dispensed for the control animals.
- Fresh dosing solutions were prepared at each concentration level weekly and dipensed for dosing daily. All dosing solutions were stored at ambient
temperature.
- Dose volume: 5 mL/kg/day
- The dose volumes were adjusted on Days 9 and 12 of gestation to changes in individual animal body weights.
VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 12, 36 and 108 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day
- Lot/batch no. (if required): 125H0892 (5 July 1997), 56H0124 (1 August 1997)
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing formulations for all three dose levels from the first three mixes used on study were assayed prior to use on study (one sample per concentration was taken and analyzed in duplicate). The formulations were considered acceptable for use on study if the two samples from each concentration level were within 10% of each other.
Dosing solutions used on study expressed as a percent of the nominal concentration for the 60, 180 and 540 mg/kg/day dose groups were 99.2%, 100.6% and 102.3%, respectively.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 ratio
- Length of cohabitation: nightly
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
Sacrifice on day 20 of gestation.
Frequency of treatment:
Single daily dose
Duration of test:
Day 6-15 of gestation
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 60, 180, and 540 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
24 females / group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Females which mated were assigned to the groups daily in such a way as to provide an equal distribution of females among groups and equalize, as best possible, the Day 0 gestation mean body weights between groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (morning and afternoon)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (morning and afternoon) for signs of pharmacologic or toxicologic effects and mortality.
- In addition, each animal was given a detailed physical examination on Days 0, 6-16, 18 and Day 20 of gestation. On Days 6-15 of gestation, the detailed physical examinations were conducted approximately one-half hour after animals had been dosed.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded on Days 0, 6, 9, 12, 16, 18 and 20 of gestation.
Day 20 gestation body weights are presented as actual and corrected (the actual Day 20 gestation body weight minus the weight of the gravid
uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Recorded for the following intervals during gestation: Days 0-6, 6-9, 9-12, 12-16 and 16-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20, exsanguination following anesthesia with carbon dioxide.
- Complete macroscopic postmortem examinations were performed on all test animals.
- External surfaces, all orifices, the cranial cavity, carcass, the external surface of the spinal cord and sectioned surfaces of the brain, nasal cavity and
paranasal sinuses, the thoracic, abdominal and pelvic cavities and their viscera and the cervical tissues and organs were examined.
- Gross lesions noted during these postmortem evaluations were retained and preserved in 10% neutral buffered formalin.
- The carcass of each female was discarded at completion of the postmortem evaluation.
- Organs Weighed: The livers were weighed for all females sacrificed on Gestation Day 20, and liver/body weight ratios were calculated using the corrected Day 20 gestation weights.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
The intact uterus (ovaries attached) was removed from the abdominal cavity, weighed and the number and location of the following were recorded for
each uterine horn:
- live fetuses (movement in response to touch);
- dead fetuses (lack of movement in response to touch with no visible degeneration);
- late resorptions (recognizable dead fetus undergoing degeneration, regardless of size);
- early resorptions (evidence of implantation but no recognizable fetus);
- implantation sites (total of fetuses plus resorptions).
- The ovaries were dissected free from the uterus and evaluated for the number of corpora lutea.
- Other: When no uterine implants were grossly apparent, the uterus was stained with ammonium sulfide (method of Salewski) to identify foci of implantation and early fetal death. When no foci were visualized poststaining, the female was considered not pregnant.
Fetal examinations:
Each fetus was individually identified, weighed, sexed externally (ano-genital distance) and given a gross examination for external malformations/variations to include observation for palatel defects.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter, staining of the ossified skeletal structures using the Alizarin Red S staining procedure of Crary. Fetal skeletal specimens were evaluated under a dissecting microscope (10-20x) for ossification variations and malformations.
- Head examinations: Yes, fetuses designated for visceral evaluation were decapitated (head placed in appropriately labelled tissue bags and fixed
in Bouin's solution for later evaluation). Following a period of fixation, the fetal heads were sectioned using a razor blade. The serial, transverse sections generated during this procedure were evaluated for malformations of the palate, eyes and brain under a dissecting microscope (10-20x)
- Late resorptions were weight, examined grossly for external malformations and discarded.
Statistics:
Statistical evaluation of equality of means was made by the appropriate one way analysis of variance technique, followed by a multiple comparison procedure if needed. First, Bartlett's test was performed to determine if groups had equal variance. If the variances were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric procedures were the standard one way ANOVA using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett's test was used to determine which means were significantly different from the control. If a nonparametric procedure for testing equality of means was needed, the Kruskal-Wallis test was used, and if differences were indicated a summed rank test (Dunn) was used to determine which treatments differed from control.
Indices:
Pre- and post-implantation loss indices, ratio of resorption to implants, Incidence of litters with Resorptions

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- Mortality
• No mortality occurred in the control group or in the groups treated with a structural analogue at dose levels of 60 and 180 mg/kg/day. All animals in these groups survived to scheduled sacrifice.
• At the 540 mg/kg/day dose level, two of 24 animals died for a mortality rate of 8.3%. Both animals died on Day 10 of gestation after 5 days of treatment. The cause of death could not be determined from the macroscopic postmortem evaluations. All other animals in this group survived to scheduled sacrifice.
- Clinical sign
• No adverse effects of treatment with structural analogue at dose levels of 60 and 180 mg/kg/day were evident from the detailed physical examinations. Excessive salivation was seen with increased frequency among the animals at the 180 mg/kg/day dose level during the treatment period. Other findings noted during the physical examination of these animals occurred at low incidence and were typical of those seen commonly in this strain of rat in this laboratory.
• At the 540 mg/kg/day dose level, the following findings were seen with increased frequency during the detailed physical examinations and considered indicative of an adverse effect of treatment: lethargy, prostration, irregular gait, excessive lacrimation, labored breathing and staining of the skin/fur in the ano-genital area. Generally, these findings were most prevalent during the Day 6-10 gestation period. Excessive salivation was also seen with increased frequency among these high-dose animals during the treatment period.
- Pregnancy Rates
• Pregnancy rates were comparable between the control and structural analogue-treated groups. Actual pregnancy rates were 91.7% (22/24), 100% (24/24), 95.8% (23/24) and 95.8% (23/24) for the 0, 60, 180, and 540 mg/kg/day dose groups, respectively.
- Body Weight Data - Gestational period
• No adverse effect of treatment with structural analogue at the 60 mg/kg/day dose level was evident from maternal body weight data during gestation. Mean weight gain for this group over the Day 6-20 gestation interval, using the corrected Day 20 gestation weights, was statistically significantly lower (-21.2 %) than control data; however, since all other maternal body weights and body weight gain data for this group were comparable to control, this difference was not considered of toxicologically significance.
• At the 180 and 540 mg/kg/day dose levels) an adverse effect of treatment was evident from maternal body weight and weight gain data. The slight, although statistically significant, decrease in mean body weight on Day 0 of gestation in the 540 mg/kg/day dose group in comparison to control data was considered fortuitous. At Day 6 of gestation, the mean body weight for this group continued to be slightly lower than control data but this difference was not statistically significant. For the remainder of the study (Days 9, 12, 16, 18 and 20 of gestation) mean body weights for the mid (- 5.8 % on gestation day 20) and high dose (-10.3 % on gestation day 20) group were lower than control data and these differences were statistically significant. Similarly, mean weight gain over the Day 6-16 gestation period, which encompassed the entire treatment period, was lower (- 22.6 %) than control in both of these groups and these differences were also statistically significant.
• At the 540 mg/kg/day dose level, animals experienced a statistically significant weight loss (-9.5 %) over the Day 6-9 gestation period and reduced weight gains (18.5 %) for the remainder of the treatment period (12-16), when compared to the control group.
• Maternal body weight gains for the 180 and 540 mg/kg/day dose groups during the post treatment period (Days 16-18, 18-20 and 16-20) were comparable to control data with the exception of the 540 mg/kg/day group which gained significantly more weight than control over days 16-18. Mean weight gains for the 180 and 540 mg/kg/day dose groups over the entire Day 6-20 gestation period using the corrected Day 20 gestation weights were lower (33.9 % and 69.9 %) than control and these differences were statistically significant.
- Food Consumption Data - Gestation Period
• No adverse effect of treatment with structural analogue was evident from maternal food consumption data during gestation at the 60 mg/kg/day dose level.
• At the 180 mg/kg/day dose level, a statistically significant reduction in food consumption was seen early in the treatment period (Days 6-9). For the remainder of the study, however, food consumption data for this group were comparable to control data.
• At the 540 mg/kg/day dose level, food consumption was lower than control throughout the treatment period and these differences were statistically significant and considered indicative of an adverse effect of treatment. During the post treatment period (Days 16-20), food consumption for the high-dose group was statistically significantly increased in comparison to control data.
- Corpora Lutea and Uterine Implantation Data
• No adverse effect of treatment with structural analogue at a dose level up to and including 540 mg/kg/day was evident from uterine implantation data. The mean numbers of corpora lutea, uterine implantations, live fetuses and resorptions per pregnant female for the treated groups were comparable to control data. Likewise, the preimplantation loss indices and the ratio of resorptions to implants for the treated groups were also comparable to control data.
- Organ weight
• Maternal Liver Weight: No adverse effect of treatment with structural analogue at a dose level of 60 mg/kg/day was seen in maternal liver weight data. As expected from the lower terminal body weights seen in the 180 and 540 mg/kg/day dose groups in comparison to control data, mean absolute liver weights for these groups were also lower than control data and these differences were statistically significant. The mean liver to body weight ratio for the 180 mg/kg/day dose group was statistically significantly lower than control-data but in the absence of a similar response in the liver to body weight ratio in the 540 mg/kg/day group, this was not considered toxicologically significant.
- Macroscopic Postmortem Evaluations
• No adverse effect of treatment with structural analogue at a dose level up to and including 540 mg/kg/day was evident from the maternal macroscopic postmortem examinations.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
ca. 60 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
ca. 180 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
180 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
- Fetal body weight:
• At dose levels up to and including 180 mg/kg bw/day, no adverse effects of treatment with structural analogue were.
• At the 540 mg/kg/day dose level, mean fetal weights (2.9 ± 0.3 g, control: 3.3 ± 0.2) distinguished by sex and as a composite for both sexes, were lower (-12 %.1 %) than control data. These differences were statistically significant and considered indicative of an adverse response to treatment.
- Fetal sex distribution data and fetal external examinations
• No adverse effect of treatment with structural analogue at a dose level up to and including 540 mg/kg/day was seen. The incidences of fetuses with external malformations for the 0 (control), 60, 180 and 540 mg/kg/day dose groups were 0.3% (1/305 fetuses), 0.6% (2/316 fetuses), 0% (313 fetuses) and 0% (300 fetuses), respectively. The incidences of litters containing fetuses with external malformations for these same groups were 4.5% (1/22 litters), 8.3% (2/24 litters), 0% (23 litters) and 0% (21 litters), respectively. These incidences on both, per fetus and per litter basis, did not differ statistically between the control and treated groups and were considered comparable between these same groups.
• The two external malformations seen among fetuses in the 60 mg/kg/day dose level were apparent absence of the lower jaw, which at skeletal examination proved to be micrognathia (small lower jaw) since a mandible was present in each instance. These external findings from these two fetuses were seen in one fetus from two different litters. Additional facial malformations (i.e., absence of a philtrum and presence of a single external nare) were also seen in the fetus of one of these two females. Similar types of jaw malformations (agnathia/micrognathia) have been seen at low incidence in the historical data of the test laboratory. In the absence of similar findings in fetuses from the higher dose levels, the occurrence of these findings in the low dose group was not considered indicative of a treatment-related response. The only other malformation seen during the fetal external examinations occurred in the control group. One control fetus had a filamentous tail. This type of malformation has been seen at low incidence in the historical control data of the test laboratory.
- Fetal Visceral Examination
• No malformations were seen during the fetal visceral examinations. A total of 158 control fetuses (22 litters), 164 low dose level (60 mg/kg/day) fetuses (24 litters), 163 mid dose group (180 mg/kg/day) fetuses (23 litters) and 155 high dose level (540 mg/kg/day) fetuses (21 litters) were evaluated. A low incidence of commonly seen visceral variations was seen in the 60 mg/kg/day group. No visceral variations were seen among the control, mid- or high-dose fetuses. The incidences of these findings on both, per fetus and per litter basis in the low-dose group were well within the range of historical control data for the test laboratory and in the absence of similar findings in fetuses from the higher dose levels, no adverse effect of treatment was indicated.
- Fetal Skeletal Examination
• No adverse effect of treatment with structural analogue at a dose level up to and including 180 mg/kg/day was seen from the fetal skeletal malformation data. No skeletal malformations were seen in the 150 fetuses (23 litters) of the mid dose group (180 mg/kg/day) or 145 fetuses (21 litters) of the high dose group (540 mg/kg/day) evaluated. Likewise, no skeletal malformations were seen in the 148 control fetuses evaluated from 22 litters. The only skeletal malformations seen during the study were in the low dose group (60 mg/kg/day). The incidence of these fetuses with skeletal malformations was 2.6% (4/153 fetuses) and the litter incidence was 16.7% (4/24 1itters). These incidences did not differ statistically from control data.
• Misshapen mandibles and misshapen cranial bones were seen in the two low dose group fetuses noted externally with jaw/facial malformations. The only other skeletal malformations seen among fetuses in this group were fused ribs and thoracic vertebral defects in one fetus from the litter of one further female of the low dose group and five lumbar vertebrae (25 presacral vertebrae) in one fetus from the litter of an additionally female of this treatment group. In the absence of similar findings in fetuses from the higher dose levels, no adverse effect of treatment was indicated from the low incidence of dissimilar skeletal malformations seen in the low-dose group.
• The incidences of fetuses with one or more ossification variations in the treated groups were comparable to or slightly lower than control data. These incidences for the control, low-, mid- and high-dose groups were 95.9%, 85.6%, 90.0% and 94.5%, respectively. The incidence of fetuses with at least one ossification for the low-dose group differed statistically from control data but this was not considered indicative of an adverse response to treatment by the authors.
• The fetal and litter incidences for the different ossification variations seen during the study for the low- and mid-dose groups were considered comparable to control data and not adversely affected by treated. Only at the 540 mg/kg/day dose level was there a suggestion of retarded fetal ossification. This was indicated from the slight increases in fetal and litter incidences of reduced ossification of the cervical vertebral transverse processes and metatarsals for this group in comparison to control data.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 540 mg/kg bw/day (actual dose received)
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
>= 180 - < 540 mg/kg bw/day (actual dose received)
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion