Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2006

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:
- Appearance: Colourless to pale yellow clear liquid
- Stability under test conditions: Stable in acetonitrile and in the mobile phase. Protect from air
- Storage conditions of test material: At room temperature protected from light
- Handling conditions of test material: Under yellow light
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: International Flavors & Fragrances (Union Beach, New Jersey, USA), Lot No. 413118

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina
- Weight at study initiation: on the day after arrical at the testing facility: males: 262-356 g, females: 188-224 g
- Housing: individual housing, except during the mating period when ech pair of male and females was housed in the male rat’s cage

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-79
- Humidity (%): 30-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing formulations were prepared weekly from bulk materials.

VEHICLE
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: 103K0107 (Sigma Aldrich)
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 analyzed for the test item content.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 / 1
- Proof of pregnancy: vaginal sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
11 days, from day 7 to day 17 of gestation (GD7 - GD17)
Frequency of treatment:
daily
Duration of test:
21 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
62 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages for this study were selected on the basis of a range finding study, in which 125, 250, 500, or 1000 mg/kg/day were administered daily on DGs 7 to 17. Adverse clinical signs occurred at 500 and 1000 mg/kg/day, and body weight gains and feed consumption were reduced during the entire dosage period at dosages of 250 mg/kg/day and higher.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included check for viability, clinical signs, abortions and premature deliveries

DETAILED CLINICAL OBSERVATIONS: Not specified

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

FOOD CONSUMPTION: Yes
- Time schedule: GDs 0, 7, 10, 12, 15, 18 and 21

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

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: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
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 analysed, 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 Cochrati 1967). Continuous data were analyzed using Bartlett's test of homogeneity of variances (Sokal and Rohlf 1969) and the analysis of variance (Snedecor and Cochran 1967), when appropriate. Dunnett's test (Dnnnett 1955) was used to idendfy Statistical significance of differences among individual groups. If the analysis of variance was not appropriate, the Kruskal-Wallis test (Sokal and Rohlf 1969) or Dunn's method of multiple comparisons (Dunn 1964) was used to identify the Statistical significance of differences among the individual groups. If there were greater than 75%, Fisher's exact test (Siegel 1956) was used to analyze the data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only clinical signs related to the test item included significantly increased (p < .01) incidences of a clear, red or yellow perioral substance and/or red perivaginal substance in the 250 mg/kg/day dosage group. Excess salivation occurred in all dosage groups, but was most common at 250 mg/kg bw/d.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains were significantly reduced (p < .01) at 250 mg/kg/day on DGs 7 to 10, whereas for the entire dosage period, body weight gain at 62, 125, and 250 mg/kg/day was 100.8%, 102.5%, and 92.4% of the control value, respectively. During the post-dosage period (DGs 18 to 21), body weight gains in all dosage groups were comparable to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Feed consumption and body weight gains were reduced at 250 mg/kg/day (Table 1). Compared to controls, mean absolute (g/day) and relative (g/kg/day) feed consumption were significantly reduced (p < .01) at 250 mg/kg/day for the entire dosage period (DGs 7 to 18), whereas at 125 mg/kg/day a significant reduction was only noted on DGs 10 to 12. For the entire dosage period, absolute feed consumption at 62, 125, and 250 mg/kg/day was 97.9%, 96.8%, and 88.4% of the control value, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross changes attributable to the test item were observed at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Three dead fetuses were observed in one litter from the 250 mg/kg/day dosage group. This observation was considered unrelated to the test item because the incidence was not statistically significant and no dead fetuses were observed in the range-finding study.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Abnormalities:
not examined

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
A significant increase (p < .05) in the percentage of live male fetuses was noted in the 125 mg/kg/day dosage group, as compared to the control group value. This finding was considered unrelated to the test item because the finding was not dose dependent and the value was within the range observed historically at the Testing Facility.
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
All fetuses appeared normal at external examination
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was only one skeletal malformaiton: a fetus in the 62 mg/kg/day dosage group had fusion of one or more ribs (right, 7th and 8th ribs medially to distally, and 9th and l0th distally), as well as skeletal variations in skull bones, ribs and sternum.
All skeletal alterations (malformations or variations) in the fetuses were considered unrelated to the test item because (1) neither the fetal nor litter incidences were dose-dependent; (2) the incidences did not significantly differ from the vehicle control group values; and/or (3) the incidences were within the ranges observed historically at the Testing Facility.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal soft tissue alterations included; an irregularly shaped brain in one 62 mg/kg/day fetus, and folded retinas, a variation usually associated with tissue processing, in 0, 2, 1, and 2 fetuses in 0,2, 1, and 2 litters in the four respective dosage groups. There were no additional alterations in these fetuses.
All soft tissue alterations (malformations or variations) in the fetuses were considered unrelated to the test item because (1) neither the fetal nor litter incidences were dose-dependent; (2) the incidences did not significantly differ from the vehicle control group values; and/or (3) the incidences were within the ranges observed historically at the Testing Facility.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: rib
visceral/soft tissue: central nervous system
visceral/soft tissue: eye

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The maternal and developmental NOAELs were determined to be 125 and > 250 mg/kg/day, respectively.
Executive summary:

The test item was evaluated for its developmental toxicity in a study equivalent to OECD guideline 414 in Sprague-Dawley rats (25/group; cesarean-sectioning identified 21 to 25 pregnant rats/group). Oral dosages of 0 (corn oil), 62, 125, or 250 mg/kg/day were administered by gavage on days 7 through 17 of gestation (GDs 7 through 17). Rats were observed for viability, clinical signs, body weights, and feed consumption. Necropsy and cesarean sectioning occurred on GD 21. Uteri were examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. Numbers of corpora lutea were also recorded. Fetuses were weighed and examined for gender, gross external changes, and soft tissue or skeletal alterations. Analysis of dosage preparations verified calculated dosages ±10%. No deaths occurred. Excessive salivation occurred in all groups, but the incidence was increased at 250 mg/kg/day. The 250 mg/kg/day dosage also was associated with a significant increase in the incidences of a clear, red or yellow perioral and/or red perivaginal substance and significant reductions in mean feed consumption and body weight gains (11.6% and 7.4%, respectively) during the entire dosage period. No gross changes attributable to the test item were observed at necropsy. Cesarean section or litter parameters, as well as fetal alterations, were not affected by the test item at 250 mg/kg/day or either of the lower dosages tested. Based on these data, maternal and developmental no-observable-effect levels (NOAELs) of 125 and > 250 mg/kg/day, respectively, were established for the test item. lt is concluded that the test item is not a developmental toxicant in rats under the conditions of this study and dosing regimen.