[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: A 28-day repeated dose study vaginal lavages to determine the stage of estrous in females were performed and spermatogenic endpoints (motility/viability, morphology, enumeration of epididymal and testicular sperm and sperm pr... (see attached file)

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

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 407 with vaginal lavages to determine the stage of estrous in females and spermatogenic endpoints (motility/viability, morphology, enumeration of epididymal and testicular sperm and sperm production rates)

Principles of method if other than guideline:

A 28-day repeated dose study vaginal lavages to determine the stage of estrous in females were performed and spermatogenic endpoints (motility/viability, morphology, enumeration of epididymal and testicular sperm and sperm production rates) were evaluated.

GLP compliance:

yes

Remarks:

OECD [C (97) 1 86/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)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: 246 g to 275 g for males; 170 g to 203 g for females
- Fasting period before study:
- Housing: housed individually in clean, wire-mesh cages suspended above cage-board, which were changed at least three times weekly.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002; ad libitum
- Water (e.g. ad libitum): Reverse osmosis-treated (on-site) drinking water; ad libitum
- Acclimation period: All animals were housed for a 14-day acclimation/pretest period.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70.4°F to 70.6°F (21.3°C to 21.5°C)
- Humidity (%): 43.2% to 51.5%
- Air changes (per hr): Air handling units were set to provide a minimum of 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark photoperiod


IN-LIFE DATES: From: 2008-08-05 To: 2008-09-02 and 03

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: PMI nutrition International, LLC Certified Rodent LabDiet® 5002

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substancewas added to PMI Nutrition International, LLC Certified Rodent 5002 on a weight/weight
basis and blended in a Hobart blender. The resulting premix was mixed thoroughly with additional PMI Nutrition International, LLC Certified Rodent 5002 in a V-Blender to obtain the appropriate dietary concentrations. Concentrations were prepared from lowest to highest. Test substance dietary admixtures will be corrected for test substance purity (correction factor of 1.159)


DIET PREPARATION
- Rate of preparation of diet (frequency): The control and test diets were prepared approximately weekly
- Mixing appropriate amounts with (Type of food): PMI nutrition International, LLC Certified Rodent LabDiet® 5002
- Storage temperature of food: placed in labeled storage bags and stored at room temperature


VEHICLE
- Justification for use and choice of vehicle (if other than water): feed in dietary study
- Concentration in vehicle: ranged from 93.2% of nominal to 107% of nominal

Details on mating procedure:

While no mating was done in this study, the assessment of multiple parameters are clearly relevant to the evaluation of reproductive organ capacity.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In the present study, diet formulations prepared at target concentrations of 300, 1000 and 10,000 ppm C-4000 were evaluated for test substance concentration acceptability.
The analyzed formulations used for test substance administration met the WIL standard operating procedure requirements for concentration acceptability for diet admix formulations, i.e., the analyzed concentrations were 85% to 115% of the target concentrations. Chromatographic analysis of the basal diet administered to the Group 1 animals contained a chromatographic peak at approximately the same retention time as the test substance and at approximately the same level as that seen in analysis of the quality control (QC) blank samples. This peak was not present in acetonitrile
injections immediately prior to injections of the QC and Group 1 formulation samples, suggesting that the peak was due to an interfering matrix component and not test substance contamination of the control diet formulation.

Duration of treatment / exposure:

28 consecutive days

Frequency of treatment:

on a continuous basis for 28 consecutive days

Details on study schedule:

No mating

Doses / concentrations

Remarks:

Doses / Concentrations:
300, 1000, and 10,000 ppm in diet
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Test substance dietary concentrations were selected by the sponsor based on the results of a prior 14-day dietary range-finding study conducted in this species and strain in which no toxicity or palatability issues were observed at test diet concentrations ≤10,000 ppm.
- Rationale for animal assignment (if not random): computerized randomization procedure

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly during the pretest period, throughout the test substance administration period and on the day prior to scheduled necropsy (non-fasted).


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; recorded weekly during the pretest period and throughout the test substance administration period. Food intake: Yes; g/animal/day for the corresponding body weight intervals
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: mean amounts of C-4000 consumed (mg/kg/day) by each sex per dose group were calculated from the mean food consumed (g/kg/day) and the appropriate target concentration of test substance in the diet (mg/kg)



OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during the pretest period (study week -1) and near the end of the treatment period (study week 3).
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes; overnight
- How many animals: all


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes; overnight
- How many animals: all


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during study week 3
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / other: physiological, home cage, handling, open field

Oestrous cyclicity (parental animals):

Vaginal lavages were performed for all female animals daily beginning on study day 14
and continuing through the day prior to necropsy.

Sperm parameters (parental animals):

The following quantitative assessments of the process of spermatogenesis were
performed for all males at the scheduled necropsy:
Motility/viability assessment
Morphology assessment
Enumeration of epididymal and testicular sperm numbers and sperm production rate

Postmortem examinations (parental animals):

SACRIFICE
- A complete necropsy was conducted for all animals. Animals were euthanized by carbon dioxide anesthesia and exsanguinated.

GROSS NECROPSY
- Gross necropsy consisted of, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities, including viscera.



HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues and organs were collected and placed in 10% neutral-buffered formalin (except as noted):

Adrenals (2)
Aorta
Bone with marrow
Femur
Sternum
Bone marrow smear (from
femur) a
Brain
Cerebrum 2 levels
Cerebellum with medulla/pons
Cervix
Epididymides (2) b
Eyes with optic nerve (2) c
Gastrointestinal tract
Esophagus
Stomach
Duodenum
Jejunum
Ileum
Cecum
Colon
Rectum
Heart
Kidneys (2)
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed by
inflation with fixative)
Lymph nodes
Mandibular (2)
Mesenteric
Nasal Cavity d
Ovaries with oviducts (2) e
Pancreas
Peripheral nerve (sciatic)
Peyer’s patches
Pituitary
Prostate
Salivary glands [mandibular (2)]
Seminal vesicles with coagulating
gland (2)
Skeletal muscle (rectus femoris)
Skin (with mammary gland)f
Spinal cord (cervical, thoracic,
lumbar)
Spleen
Testes (2) b
Thymus
Thyroid [with parathyroids, if
present (2)]e
Trachea
Urinary bladder
Uterus
Vagina
Gross lesions (when possible)

a - Not taken from animals found dead, not placed in formalin; not examined.
b - Testis and epididymis (right only) placed in Bouin’s solution.
c - Fixed in Davidson’s solution
d - Levels I and III according to method of Young (Young, 1981) examined
e - Parathyroids and oviducts examined microscopically if in plane of section or if gross lesion present.
f - For females; a corresponding section of skin was taken from the same anatomic area for males.

The following organs were weighed from all animals at the scheduled necropsy:
Adrenals
Brain
Epididymides (total and cauda)*
Heart
Kidneys
Liver
Ovaries with oviducts
Spleen
Testes*
Thymus

Paired organs were weighed together. Designated organs (*) were weighed separately.

Statistics:

All statistical tests were performed using appropriate computing devices or programs.
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance level 5%, comparing each test substance-treated group to the control group by sex. 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. Statistical analyses were not conducted if the number of animals was 2 or less. Due to the use of significant
figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not specified

Details on results (P0)

CLINICAL SIGNS AND MORTALITY- unaffected


BODY WEIGHT AND WEIGHT GAIN- unaffected


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)- unaffected



OPHTHALMOSCOPIC EXAMINATION- unaffected


HAEMATOLOGY- unaffected


CLINICAL CHEMISTRY- higher total cholesterol in 10,000 ppm males



NEUROBEHAVIOUR- unaffected


ORGAN WEIGHTS- group mean absolute relative to body liver weights were slightly higher at 10,000 ppm; statistically significant only on a relative basis


GROSS PATHOLOGY- considered to be spontaneous and/or incidental in nature and unrelated to test substance administration


HISTOPATHOLOGY: limited to the liver and thyroid of the 10,000 ppm group males and females:
Microscopic examination of the liver revealed minimal enlargement of centrilobular hepatocytes due to cytoplasmic hypertrophy of a few 10,000 ppm group male and female rats. Minimal or mild hypertrophy and hyperplasia of the follicular epithelial cells of the thyroid was observed in animals of all groups, including the controls, with an increased incidence in the 10,000 ppm group males and females.


OTHER FINDINGS-It is relevant to the evaluations of selected reproductive organ parameters conducted in this study [estrous cycle evaluation in females, and epididymal sperm count, sperm production rate, motility and the percentage of morphologically normal sperm in males] that there were no test substance-related microscopic findings in reproductive organs of males or females in the 300, 1,000 or 10,000 ppm groups.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

10 000 ppm

Sex:

male/female

Basis for effect level:

other: overall effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

No mating was done in this study

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

All physiological parameters assessed in this study [estrous cycle evaluation in females, and epididymal sperm count, sperm production rate, motility and the percentage of morphologically normal sperm in males] plus the correlative microscopic findings in reproductive organs of males and females indicated that there were no treatment-related effects on the reproductive organs of males or females in the 300, 1,000 or 10,000 ppm groups.

The results of this study strongly suggests that oral dietary administration of C-4000 at concentration of 10,000 ppm [approximately 723 mg/kg/day for males and 898 mg/kg/day for females] for 28 days is a reproductive NOEL.

Executive summary:

A 28-day repeated dose study vaginal lavages to determine the stage of estrous in females were performed and spermatogenic endpoints in males (motility/viability, morphology, enumeration of epididymal and testicular sperm and sperm production rates) were evaluated.

No adverse effects were observed even at the highest doses of 23 mg/kg/day for males and 898 mg/kg/day for females. There were no signs of mortality or clinical manifestations.