[No public or meaningful name is available]

Developmental toxicity / teratogenicity

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: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

States that animals would be observed approximately 1 hour following dose administration. Female nos. 29934 and 29937 in the 180 and 1000 mg/kg/day groups, respectively, were observed 1 hour 21 minutes and 1 hour 16 minutes, respectively, following dose a

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Remarks:

US EPA 40 CFR Parts 160 and 792; OECD [C(97) 186/Final]

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): C-4000
- Physical state: liquid
- Analytical purity: 86.3 +/- 0.7%
- Purity test date: 2008-07-09
- Lot/batch No.: 51V034K7
- Expiration date of the lot/batch: 2008-10
- Storage condition of test material: refrigerated

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: approximately 12 weeks old when paired for breeding
- Weight at study initiation: 224 to 278 g on gestation day 0
- Fasting period before study: none
- Housing: Upon arrival and until pairing, all rats were individually housed in clean, stainless steel wire-mesh cages suspended above cage-board. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were returned to individual suspended wire-mesh cages
- Diet (e.g. ad libitum): basal diet, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, ad libitum
- Water (e.g. ad libitum):Reverse osmosis-purified (on-site) drinking water, ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70.2°F to 72.1°F (21.2°C to 22.3°C)
- Humidity (%): 39.3% to 46.6%
- Air changes (per hr): a minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): a 12-hour light/12-hour dark photoperiod


IN-LIFE DATES: From: 2008-10-16 (gestation day 0) To: 2008-11-24 (experimental termination)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance formulations were weight/volume (test substance/vehicle) mixtures. Formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensing and stored refrigerated (2-8ºC).
The test substance formulations were stirred continuously throughout the preparation, sampling and dose administration procedures. The first dosing formulations were visually inspected by the study director and were found to be visibly homogeneous and acceptable for administration.



VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 6, 36 and 200 mg/mL (correction factor of 1.159)
- Amount of vehicle (if gavage):
- Lot/batch no. (if required): 028K0044 and 117K0127,

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

High performance liquid chromatography (HPLC) analytical method which uses ultraviolet (UV) absorbance detection at a wavelength of 235 nm to measure C-4000 concentration in corn oil formulations ranging in concentration from 4.00 to 200 mg/mL.

Duplicate formulation samples were collected using a syringe and dosing cannula and placed in polypropylene tubes. As detailed in the following table, formulation samples were processed by adding ACN and mixing by vortex action. The samples were further diluted with ACN as necessary to achieve a theoretical final concentration of 100 µg C-4000/mL. Aliquots of the diluted samples were transferred to amber autosampler vials for analysis.

The HPLC/UV method used to determine C-4000 concentration in corn oil formulations was validated for the concentration range from 4.00 to 200 mg/mL. Dose formulations of C-4000 in corn oil, prepared at target concentrations of 6, 36 and 200 mg/mL ranged from 94.8% to 103% of nominal, and met the WIL SOP acceptance criteria for test substance concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Following positive evidence of mating
- Verification of same strain and source of both sexes: yes; resident male from the same strain and source for breeding
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

gestation days 6 through 19

Frequency of treatment:

once daily

Duration of test:

2008-10-02 (animal receipt date) through 2008-11-24 (experimental termination)

Doses / concentrations

Remarks:

Doses / Concentrations:
30, 180 and 1000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the absence of maternal or developmental toxicity in the dose range-finding study and the presence of minimal test substance-related microscopic findings at a dosage level of 10000 ppm [approximately 898 mg/kg/day in females] in the 28-day oral dietary rat
study, dosage levels of 30, 180 and 1000 mg/kg/day were selected for this developmental toxicity study.
- Rationale for animal assignment (if not random): computerized randomization

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: recorded from gestation days 0 through 20 (prior to dose administration during the treatment period).
Animals were also observed for signs of toxicity approximately 1 hour following dose administration.


BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0 and 6-20; daily


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: gestation days 0 and 6-20; daily



POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: uterus and ovaries


OTHER: placenta

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: placenta

Fetal examinations:

- External examinations: Yes: all viable fetuses
- Soft tissue examinations: Yes: heads of one half of the fetuses fromeach litter
- Skeletal examinations: Yes: all fetuses
- Head examinations: Yes: one half of the fetuses from each litter

Statistics:

All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherwise) for a minimum significance level of 5%, comparing each test substance-treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.) and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the different rounding conventions inherent in the types of software used, the means, standard deviations and standard errors on the summary and individual tables may differ by ±1 in the last significant figure. Where applicable, the litter was used as the experimental unit.

Mean maternal body weights (absolute and net), body weight changes (absolute and net) and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal and combined) and each particular
external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.

Indices:

1. Group Mean Litter Basis:

Postimplantation Loss/Litter = No. Dead Fetuses, Resorptions (Early/Late)/Group
____________________________________________
No. Gravid Females/Group

2. Proportional Litter Basis:

Summation Per Group (%) = Sum of Postimplantation Loss/Litter (%)
___________________________________
No. Litters/Group

Where, Postimplantation Loss/Litter (%) = No. Dead Fetuses, Resorptions (Early/Late)/Litter
_____________________________________________ x 100
No. Implantation Sites/Litter

Historical control data:

WIL Developmental Historical Control Data Version 3.8 [Crl:CD(SD) Rats]

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Oral administration of the test material, C-4000, by gavage to pregnant Crl:CD(SD) rats produced no evidence of treatment-related developmental toxicity or maternal toxicity at any dose in this study. A dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was the no-observed-adverse-effect level (NOAEL) for maternal and prenatal developmental toxicity.

Executive summary:

The objective of the study was to determine the potential of C-4000 to induce developmental toxicity after maternal exposure via the oral route during the critical period of organogenesis, to characterize maternal toxicity at the exposure levels tested and to determine the no-observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity.

The test substance, C-4000, in the vehicle, corn oil, was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily on gestation days 6 through 19. Dosage levels were 30, 180 and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. The doses were prepared using a correction factor for purity (1.159). A concurrent control group of 25 females received the vehicle (corn oil) on an identical regimen. The females were approximately 13 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each surviving female. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.

One female in the control group was euthanized in extremis following body weight loss and minimal food consumption. All other females survived to the scheduled necropsy; there were no test substance-related internal findings at any dosage level. Additionally, there were no adverse clinical findings noted for females in any group at the daily examinations, at the time of dosing or approximately 1 hour following dose administration. Mean maternal body weights, body weight gains and food consumption were unaffected by test substance administration. Intrauterine growth and survival of fetuses were unaffected by test substance administration at all dosage levels. No test substance-related fetal morphological malformations or developmental variations were noted for any fetus in this study.

Oral administration of the test material, C-4000, by gavage to pregnant Crl:CD(SD) rats produced no evidence of treatment-related developmental toxicity or maternal toxicity at any dose in this study. A dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was the no-observed-adverse-effect level (NOAEL) for maternal and prenatal developmental toxicity.