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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: Acceptable, well-documented study performed according to appropriate regulatory guidelines, GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MRD-94-825
- Substance type: liquid
- Analytical purity: assumed 100%

Test animals

Species:
rat
Strain:
other: Crl:CD BR VAF/Plus
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: Approximately 11-13 weeks
- Weight at study initiation: Females: 203 to 295 grams,
- Fasting period before study: n/a
- Housing: Single housed during the study period, except during mating.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:21 days; animals were checked for viability at least once daily.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24.4 degrees Celsius
- Humidity (%): 40 to 70 percent relative humidity
- Air changes (per hr): n/a
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
mixing

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: room
Analytical verification of doses or concentrations:
no
Details on mating procedure:
At approximately 3:00 p.m., on the initial scheduled mating day, females were placed in males' cages in a 1:1 (male: female) ratio. Males and females were paired based on sequential Physical Identification Number. A suitable number of animals were co-housed in an attempt to produce a suitable number of mated animals to accommodate lab scheduling. Mating was confirmed on the following morning by observation of a copulatory plug (vaginal) and/or by the presence of sperm in a vaginal rinse. The day on which mating was confirmed was the female's Day 0 of gestation (GD 0). After confirmation of mating, each mated female was returned to its own cage. New females then were placed in the males' cages until the required number of mated females was obtained by continuous cohabitation in consideration of lab scheduling. Mated females were assigned to dose groups in the order of mating. Accordingly, the first confirmed mated female was assigned to Group 1, the next to Group 2, and so on until all mated animals for a given day were assigned to dose groups. On subsequent days, the next group in sequence was filled by the first confirmed mated female on that day, and so on. Assignments were made until all groups were filled with confirmed mated females.
Duration of treatment / exposure:
Gestation day 6 through 15
Frequency of treatment:
Once daily
No. of animals per sex per dose:
25
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: from previous range-finding study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes:
- Soft tissue examinations: Yes
- Skeletal examinations: Yes:
- Head examinations: Yes:
Statistics:
Statistical treatment of the results was conducted where appropriate. Statistical evaluation of equality of means was done by an appropriate one way analysis of variance and a test for ordered response in the dose groups. First, Bartlett's Test was performed to determine if the dose groups had equal variance. If the variances were equal, the testing was done using parametric methods, otherwise nonparametric techniques were used.

Where applicable, percentages were calculated and transformed by Cochran's transformation, followed by the arc sine transformation. The raw percentages and the transformed percentages both were tested for statistical significance. Transformations were not reported since they were not statistically significant.

For the parametric procedures, a standard one way ANOVA using the F distribution to assess significance was used. If significant differences among the means were indicated, Dunnett's Test was used to determine which treatment groups differed significantly from control. In addition to the ANOVA, a standard regression analysis for linear response in the dose groups was performed. The regression also tested for linear lack of fit in the model.

For the nonparametric procedures, the test of equality of means was performed using the Kruskal-Wallis Test. If significant differences among the means were indicated, Dunn's Summed Rank Test was used to determine which treatment groups differed significantly from the control. In addition to the Kruskal-Wallis Test, Jonckheere's Test for monotonic trend in the dose response was performed.

Bartlett's Test for equal variance was conducted at the 1% level of significance. All other tests were conducted at the 5% and 1% level of significance.
Indices:
N/A
Historical control data:
N/A

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Adverse clinical signs, reductions in body weight gain and food consumption

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
No adverse effects.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
other: maternal dose
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Differences in the incidence of fetal skeletal variations were observed in the treated groups compared with controls. Statistically significant increases in fetal variations from controls were as follows:

                FETAL

           PARAMETER

DOSE

(MG/KG)

FETAL

INCIDENCE (%)

LITTER

INCIDENCE (%)

HISTORICAL CONTROL RANGE (%)

TOTAL WITH SKELETAL VARIATIONS

0

100

500

1000

33.7 

24.3 

43.5  

61.0**

75.0

75.0

91.7

 94.7 

16.8 - 57.8 F

 

62.5 - 100.0 L

RUDIMENTARY LUMBAR RIBS

0

100

500

1000

23.2 

17.3 

31.4 

39.0**

62.5 

54.2 

91.7* 

73.7 

3.7 - 21.6 F

 

25.0 - 61.9 L

WELL FORMED LUMBAR RIBS

0

100

500

1000

 0.0

 0.6

  3.7*

 4.8**

 0.0

 4.2

16.7

 26.3*

0.0 F

 

0.0 L

SKULL BONE(S) HYPOPLASTIC

0

100

500

1000

7.7 

2.9 

8.4 

19.2**

29.2

16.7

37.5

52.6

0.0 - 17.65 F (A)

 

0.0 - 54.17 L (A)

There was a statistically significant increased incidence of total skeletal variations in the high dose group compared with controls on a per fetus basis. However, this incidence (61%) was only slightly higher than the historical control range of this laboratory (16.8-57.8%). Additionally, since the litter is the preferred unit of measure in developmental toxicology, the absence of a statistically significant increase in the litter-based incidence of total fetal variations provides additional evidence the observed increase in this parameter was not biologically meaningful. Additionally, the litter-based incidences were within historical controls range for this laboratory.

Applicant's summary and conclusion

Conclusions:
Exxal 8N is not a developmental toxin at a dose of 1000 mg/kg/day during gestation day 6-15.
Executive summary:

This study was conducted to evaluate the potential developmental toxicity of EXXAL 8N. The test material was administered by gavage to three groups of Crl:CDBR female rats at doses of 100, 500, and 1000 mg/kg. A fourth group (Group 1) served as a control and received the carrier (corn oil) only. Mated females were dosed once daily from Gestation Day (GD) 6 through GD 15. Dosing volumes were 5 ml/kg for all groups and based on the animals' most recent body weight.

 

Males and females were paired and housed overnight until confirmation of mating (sperm/plug = GD 0). Each mated female then was returned to its own cage and new females were placed in the males' cages until the required number of mated females was obtained. Mated females were assigned to dose groups in the order of mating.

 

Clinical observations were made daily during gestation. The animals were examined for viability at least twice daily during the treatment period and at least once daily at other times during the study. Body weight and food consumption measurements were made on GD 0, 6, 9, 12, 15, 18, and 21. On GD 21, animals were euthanized and cesarean sections were performed. Gross necropsies were performed, uterine weights with ovaries attached measured, uterine contents examined, and the required uterine implantation data recorded. All live fetuses were weighed, sexed externally, and examined externally for gross malformations. 

 

Approximately one-half of the fetuses of each litter were decapitated after being euthanized. The heads were preserved in Bouin's solution for at least two weeks, rinsed, and subsequently stored in 70% alcohol. Sections of the fetal heads were prepared with a razor blade, examined for the presence of abnormalities, and then discarded. The viscera of these fetuses were immediately examined for abnormalities by dissection. The remaining live fetuses were eviscerated, processed for skeletal staining, and examined for the presence of malformations and ossification variations.

 

Unscheduled mortality was limited to one high dose female which was euthanized in a moribund condition on GD 9. This animal was observed with extreme abdominal staining just prior to death. There were no significant findings at postmortem examination and therefore, the cause of morbidity could not be established. 

 

Adverse clinical signs were observed primarily in the high dose dams following dose initiation. Approximately eight of the twenty-four surviving dams were observed with a least one of the ensuing signs: emaciation, little sign of food consumption, abdominal/anogenital staining, rales, hypoactivity, no stool, and/or little sign of stool. These signs were transient, occurring for only one day in some animals, and were generally not observed in the animals following the cessation of dosing. The remaining dams in the group were free of observable abnormalities throughout the study, or had incidental findings such as alopecia. The majority of dams in the control, low and mid dose groups were free of observable abnormalities during the entire gestation period. 

 

Statistically significant body weight gain suppression was observed in the high dose females at the GD 6-9 body weight change interval and during the overall treatment period (GD 6-15) when compared with controls. Statistically significant decreases in mean food consumption were observed in the high dose females during the GD 6-9 interval and during the treatment period (GD 6-15) when compared with controls. However, these changes were transient and corrected mean body weight and mean food consumption of all treated group females were essentially equivalent for the overall gestation period (GD 0-21). 

 

There were no maternal findings found postmortem which were judged to be the result of treatment with EXXAL 8N. The majority of uterine implantation parameters were essentially equivalent between treated and control groups. There was an increase in post-implantation loss in the high dose females (17.3%) compared with controls (4.8%), although this difference was not statistically significant. Three dams in the high dose group did not have any viable fetuses at cesarean section, and only had implantation sites or early resorptions. The resorption of these fetuses was considered the result of the poor health of the dams rather than a direct developmental effect.

 

There were no statistically significant differences in mean fetal body weight between treated and control fetuses of either sex. Three low dose, two mid dose, and one high dose fetus were stunted. There were no statistically significant increases in total or individual external, visceral, or skeletal malformations, or external and visceral variations in the treated groups when compared with controls. Additionally, there were no statistically significant differences in the mean skeletal ossification sites between treated and control fetuses. 

 

There were statistically significant increases in total fetuses with skeletal variations and in the incidence of hypoplastic skull bones in the high dose group when compared with controls. However, the incidences of these findings were only slightly higher than the historical control range of this laboratory. Additionally, since the litter is the preferred unit of measure in developmental toxicology, the absence of a statistically significant increase in the litter-based incidence provides additional evidence the observed increase in this parameter was not biologically meaningful.

 

Statistically significant increases in rudimentary and well formed lumbar ribs were observed in the high and/or mid dose groups compared with controls on a per fetus and/or litter basis. On a per litter basis the increase in rudimentary lumbar ribs was statistically significant in the mid dose group when compared with controls. Although these increased incidences were not within the historical range of this laboratory, variations in skeletal structure such as rudimentary ribs are so common, they are regarded as alternative normal patterns and not regarded as harmful developmental toxic effects. Furthermore, rudimentary or well formed ribs are not considered biologically significant in the absence of other conventional signs of embryotoxicity, i.e. malformations, embryolethality, or fetal weight reduction. Rib variations have often been associated with maternal toxicity or stress. Therefore, the developmental significance of rib variations in the high dose fetuses probably relates to the transient maternal toxicity observed in the high dose females during the treatment phase of this study. Thus, this variation was not considered to be an adverse effect on the fetuses.  

 

In conclusion, signs of toxicity were apparent in the maternal animals at a dose level of 1000 mg/kg, as indicated by adverse clinical signs, reductions in body weight gain and food consumption. However, the findings were generally transient. There also was an increase in post-implantation loss in the high dose females, as three dams did not have any viable fetuses at cesarean section, only implantation sites/early resorptions. The resorption of these fetuses was considered the result of the poor health of the dams rather than a direct developmental effect. There were several statistically significant increases in the incidence of fetal skeletal variations both on an individual and litter basis in the treated groups compared with controls. However, most of these incidences were only slightly higher than the historical control range of this laboratory, or regarded as alternative normal patterns, and not regarded as harmful developmental toxic effects (Harris and DeSesso, 1994). Therefore, these common findings in fetal rats were not considered biologically important. Accordingly, the maternal NOAEL (No Observable Adverse Effect Level) was established at 500 mg/kg. The developmental NOAEL was established as 1000 mg/kg under the conditions of this study.