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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
The requirements of the U.S. Food and Drug Administration (FDA)(1) were used as the
basis for study design.
Principles of method if other than guideline:
The study was designed to evaluate ICH Harmonised Tripartite Guideline states C and D of the reproductive process.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Sample No.: 929473

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)IGS BR VAF/Plus
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 213 - 241 g
- Housing: in compliance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources 1996), individual housing on the basis of computer-generated random units,
- Diet (e.g. ad libitum): ad libitum, Certified Rodent Diet® 5002 (PMI Nutrition International, St. Louis, MO)
- Water (e.g. ad libitum): ad libitum, reverse osmosis deionized water (with chlorine added to the processed water as a bacteriostat)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26 (64 - 79 F)
- Humidity (%): 30 - 70
- Air changes (per hr): >=10 changes per hour of 100 % fresh air passed through 99.97 % HEPA filters
- Photoperiod (hrs dark / hrs light): 12 / 12

OTHER
- healthy mated females were assigned to four dosage groups, 25 rats/group, using a computer-generated (weight-ordered) randomized procedure based on body weights recorded on the day when sperm was found in the vaginal smear or a copulatory plug was found in the vagina

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- dosing formulations were prepared daily from bulk materials
- dosage volume of 10 ml/kg bw was adjusted daily according to individual body weights recorded directly before gavage and was administered at approximately the same time each day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from each concentration of the dosing suspensions (first and last days of treatment) were analysed for alpha-iso-methylionone (AIM) content by International Flavors and Fragrances, Inc.
Details on mating procedure:
- 5-day mating period
- each pair of male and female rats was housed in the male rat's cage
- the presence of spermatozoa and/or a copulatory plug was designated as gestational day 0 (GD 0)
Duration of treatment / exposure:
10 days (GD 7 to 17)
Frequency of treatment:
daily
Duration of test:
21 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 pregnant females/group in definitive developmental toxicity study
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were selected on the basis of the following range-finding study.
- Rationale for animal assignment (if not random): All rats were pregnant and all caesarean sectioning and litter observations were comparable among the five groups. No fetal gross external alterations were observed. Based on these data, a dosage greater than 10 mg/kg/day AIM was recommended for the definitive developmental toxicity study in rats.

Examinations

Maternal examinations:
- Body weights were recorded prior to the start of the study and daily during the dosage and postdosage periods. Feed consumption was recorded on GDs 0, 7, 10, 12, 15, 18, and 21.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes, also for abortions, and premature deliveries
- Time schedule: before dosage administration and approximately 1 h later


BODY WEIGHT: Yes
- Time schedule for examinations:prior to the start of the study and daily during the dosage and postdosage periods


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, on GDs 0, 7, 10, 12, 15, 18, and 21

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21, by inhalation of carbon dioxide
- Organs examined: gross necropsy of the thoracic, abdominal, and pelvic viscera was performed

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
- Other: Uteri of apparently nonpregnant rats were examined while pressed between glass plates to confirm the absence of implantation sites.
Fetal examinations:
- Fetuses were removed from the uterus, weighed, and examined for gender and gross external alterations.
- Live fetuses then were euthanized by an intraperitoneal injection of pentobarbital before undergoing further examination.
- Approximately half of the fetuses in each litter were fixed in Bouin’s solution and examined for soft tissue alterations, using a variation of Wilson’s sectioning technique (Staples 1974; Wilson 1965).
- The remaining fetuses in each litter were eviscerated, cleared, stained with alizarin red S (Staples and Schnell 1964), and examined for skeletal alterations.
Statistics:
- Data generated during the course of study were recorded either by hand or using the Argus Automated Data Collection
and Management System and the Vivarium Temperature and Relative Humidity Monitoring System. All data were tabulated,
summarized, and/or statistically analyzed using the above systems in conjunction with Microsoft Excel (Microsoft Office 97,
version SR-2) and/or The SAS System (version 6.12).
- Clinical observation and other proportion data were analyzed using the variance test for homogeneity of the binomial distribution
(Snedecor and Cochran 1967a).
- Continuous data were analyzed by using Bartlett’s test of homogeneity of variances (Sokal and Rohlf 1969a) and the analysis of variance (Snedecor and Cochran 1967b).
- Dunnett’s test (Dunnett 1955) was used to identify statistical significance of individual groups.
- If the analysis of variance was not appropriate, the Kruskal-Wallis test (Sokal and Rohlf 1969b) or Dunn’s method of multiple comparisons (Dunn 1964) was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s exact test (Siegel 1956) was used to analyze the data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
see below

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
> 30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity (no effects observed)
Remarks on result:
other: highest dose tested

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
see below

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed in the fetuses

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

range finding study:

- Food consumption during the dosage period was comparable among the five groups.

- Although not dosage dependent, body weight increased during the dosage period in the 1.25, 2.5, 5, and 10 mg/kg/day AIM-treated rats were 117.6%, 111.4%, 119.4%, and 111.4% of the vehicle control values.

- During the postdosage period, increase in gain of body weight remained, respectively.

- All rats were pregnant and all caesarean sectioning and litter observations were comparable among the five groups.

Definitive developmental toxicity study:

maternal findings:

- No mortality occurred during the study.

- Clinical signs that included occasional incidences of chromorhinorrhea, excess salivation, sparse hair coat, localized alopecia, urine-stained abdominal fur, soft or liquid feces, or decreased activity were observed in a few animals from each group of rats. None of these clinical signs were attributed to AIM because the number of affected rats was not dosage related and did not significantly differ between control and treated groups.

- Food consumption and body weight gains were unaffected by dosages of AIM as high as 30 mg/kg/day (Table 1). For the dosage period, absolute food consumption at 3, 10, and 30 mg/kg/day was 101.0%, 102.1%, and 102.6% of the control value, respectively, whereas body weight gains were 99.0%, 99.9%, and 101.2% of the control value over the same time period. Body weight gains were also comparable during the post- dosage period (GDs 18 to 21).

Table 1:

  Dosage group (mg/kg bw/day)
   0 (control)  3  10  30
 rats tested  25  25  25  25

 rats pregnant

 24  25  24  21
 maternal absolute food consumption in g/day (mean ± SD)     
GDs 0 - 7   23.0 ± 2.4  22.9 ± 1.9 23.5 ± 1.7   23.1 ± 2.1
GDs 7 - 10  18.6 ± 3.2  18.6 ± 1.8  19.2 ± 2.9   19.3 ± 3.2
GDs 7 - 18   19.4 ± 2.4 19.6 ± 1.8  19.8 ± 2.2  19.9 ± 2.1 
GDs 18 - 21   26.4 ± 2.8 26.0 ± 2.6   27.4 ± 2.2 26.0 ± 2.1 
GDs 7 - 21   21.0 ± 2.3 21.2 ± 1.7   21.5 ± 2.0 21.2 ± 1.9 
 GDs 0 - 21  21.7 ± 2.1 21.8 ± 1.4  22.2 ± 1.7  21.8 ± 1.8 

 maternal body weight gains in g (mean ± SD)         

 
 GDs 0 - 7 36.5 ± 7.4  33.1 ± 7.6  37.9 ± 5.4  34.2 ± 9.2 
 GDs 7 - 10  11.2 ± 5.9 12.9 ± 4.2  13.8 ± 7.6  15.5 ± 7.0 
 GDs 7 - 18 82.4 ± 12.1  81.6 ± 13.3   82.3 ± 15.3  83.4 ± 12.3
 GDs 18 - 21 58.2 ± 9.0  58.6 ± 8.1  55.5 ± 20.2   54.9 ± 8.0
 GDs 7 - 21 140.5 ± 14.5   140.2 ± 16.4 137.8 ± 30.2   138.3 ± 15.8
 GDs 0 - 21  177.0 ± 18.1 173.2 ± 21.3  175.8 ± 33.1  172.6 ± 17.8 

- Pregnancy occurred in 24 (96%), 25 (100%), 24 (96%), and 21 (84%) of the 25 rats in the 0, 3, 10, and 30 mg/kg/day dosage groups, an event determined before exposure was initiated.

- Number of caesarean sectioning or litter parameters were not affected by dosages of AIM at 30 mg/kg/day (Table 2).

- The litter averages for corpora lutea, implantations, litter sizes, live fetuses, resorptions, fetal body weights, percentage of dead or resorbed conceptuses, and percentage of live male fetuses were comparable among the three dosage groups, did not significantly differ from the vehicle control group values, and were within the ranges observed historically at the Testing Facility.

Table 2:

Dosage group (mg/kg bw/day)  0 (control)  3  10  30
 Rats tested  25  25  25  25
Rats caesarean sectioned (%)   24 (96)  25 (100)  24 (96)  21 (84)
Corpora lutea / rat (mean ± SD)  15.3 ± 1.8  16.3 ± 2.3   16.0 ± 2.1 16.7 ± 2.3 
 Implantations / rat (mean ± SD)  15.0 ± 1.6 14.9 ± 1.8  14.5 ± 1.4  14.7 ± 1.8 
 Resorptions / rat (mean ± SD) 0.2 ± 0.5  0.4 ± 0.6   0.5 ± 0.6 0.4 ± 0.6 
 Litter size / rat (mean ± SD)  14.7 ± 1.7 14.5 ± 1.8  14.0 ± 1.4   14.3 ± 1.6
 Fetal body weight / litter (mean ± SD) 5.31 ± 0.29   5.20 ± 0.34  5.26 ± 0.64 5.47 ± 0.26 
 Live fetuses / group  353  363  336  301
 Dead fetuses / group  0  0  0  0

fetal findings:

- Fetal evaluations were based on 353, 363, 336, and 301 live caesarean-delivered fetuses from the 0, 3, 10, and 30 mg/kg/day dosage groups, respectively.

- Each of these fetuses was examined for gross external alterations and soft tissue or skeletal alterations (see Table 3 for detailed analyses). Fetal alterations were defined as either malformations (irreversible changes that occur at low incidences with this species and strain) or variations (common findings in this species and strain including reversible delays or accelerations in development).

- The only fetal gross external alterations occurred in one control fetus in which a depressed left eye bulge, a fleshy thoracic protrusion, and the presence of an extra hind limb were observed. Skeletal examination of this fetus revealed a small left eye socket; the presence of three femurs, tibia, and fibulas on the right side of the body (partially fused), and extra metatarsals, digits, and three sets of phalanges (four, five, and five) on the right hindlimbs.

- No soft tissue malformations occurred in any of the groups. Soft tissue variations were limited to folded retinas in seven fetuses from two different litters at 10 mg/kg/day (variations considered to be artifacts of processing), slight dilation of the renal pelvis in one fetus from the control and one fetus from the 3 mg/kg/day dosage groups, and an aberrant umbilical artery in one and two fetuses at 3 and 10 mg/kg/day, respectively.

- Skeletal malformations were absent (except in the control fetus already described), and skeletal variations were limited to infrequent incidences of delayed ossification at various bone ossification centers (non–dose dependent) and the presence of cervical ribs at the 7th cervical vertebra in four fetuses in each of the control and 30 mg/kg/day dosage groups.

- All fetal gross external, soft tissue, or skeletal alterations (malformations or variations) were considered unrelated to AIM because (1) neither the fetal nor litter incidences were dosage dependent;

(2) the incidences did not significantly differ from the control group values; and/or

(3) the incidences were within the ranges observed historically at the Testing Facility.

Table 3:

 Dosage group (mg/kg bw/day)  0 (control)  3  10  30
 Litters evaluated  24  25  24  21
 Fetuses evaluated  353  363  336  301
 Litters with fetuses with any observed alterations (%)  5 (10.8)  7 (28.0)  7 (29.2)  2 (9.5)
 Fetuses with any observed alterations (%)  6 (1.7)  7 (1.9)  14* (4.2)  5 (1.7)
 % fetuses / litter with any alterations (mean ± SD)  1.6 ± 3.4  1.9 ± 3.1  3.8 ± 7.6  1.8 ± 6.8

* includes folded retinas in three and four fetuses in two separate litters, findings considered to be artifacts of processing.

Applicant's summary and conclusion

Conclusions:
In this pre-natal development toxicity studies in female rats, the NOAEL for maternal toxicity as well as developmental toxicity was found to be above the highest dose tested of 30 mg/kg bw/day.
Executive summary:

A developmental study was conducted on 100 (25/dose) presumed pregnant female rats. alpha-Iso-methylionone was administered by gavage on days 7-17 of gestation at dose levels of 0, 3, 10 or 30 mg/kg/day in corn oil. The animals were observed twice daily for mortality and morbidity. Clinical observations of test material effects and observations for abortion and premature delivery were conducted before and approximately one hour following dosing and once daily thereafter. Bodyweights were recorded prior to the start of the study and daily during dosage and post dosage periods. Feed consumption was recorded on days 0, 7, 10, 12, 15, 18 and 21. On day 21, all rats were sacrificed, caesarean sectioned and a gross necropsy was conducted on all animals. The number and distribution of corpora lutea were recorded. The uterus of each rat was removed and examined for pregnancy, number and distribution of implantation, fetal mortality and early and late resorptions. All female rats survived to scheduled sacrifice. All clinical and necropsy observations were considered to be unrelated to the administration of the test material. Maternal body weights, bodyweight gains and absolute and relative feed consumption values were unaffected at dosages of test material as high as 30 mg/kg/day. Pregnancy occurred in 21 of 25 rats in each dosage group. Caesarean-sectioning and litter parameters were not affected by dosages of the test material as high as 30 mg/kg/day. No fetal alterations occurred that were considered associated with the test material. It was concluded that the maternal and developmental no-observable-adverse-effect levels (NOAEL) for alpha-iso-methylionone were greater than 30 mg/kg/day.