[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

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 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050

GLP compliance:

yes

Remarks:

US EPA 40 CFR Part 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: refrigeratede

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 oral 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

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

Doses / concentrations

Remarks:

Doses / Concentrations:
300, 1000 and 10000 ppm
Basis:
other: active ingredient; prepared using a correction factor for purity (1.159)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: doses determined based on the results of a 14-day range finding study
- Rationale for animal assignment (if not random): computerized randomization

Examinations

Observations and examinations performed and frequency:

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


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal and
pelvic cavities, including viscera.
Macroscopic Examination
A gross necropsy was conducted on all animals dying spontaneously, euthanized in extremis or at the scheduled necropsy. Animals euthanized in extremis or at the study termination were euthanized with C02 and exsanguinated. Necropsy included examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities, including viscera. At the time of necropsy the following tissues and organs were collected and placed in 10% neutral-buffered formalin (expect as noted).
Adrenals (2)
Lymph nodes
Aorta
Mesenteric
Bone with marrow
Sternum
Femur
Mandibular
Nasal cavity(d)
Ovaries (2) with oviducts(e)
Bone marrow smear (from femur[a])
Pancreas
Brain
Cerebrum Level 1
Cerebrum Level 2
Cerebellum with medulla/pons
Peripheral nerve (sciatic)
Peyer's patches
Pituitary
Prostate
Cervix
Salivary glands [mandibular (2)]
Epididymides (2)(b)
Seminal vesicles with coagulating gland (2)
Eyes with optic nerve (2)(c)
Gastrointestinal tract
Esophagus
Stomach
Duodenum
Jejunum
Ileum
Cecum
Colon
Rectum
Skeletal muscle (rectus femoris)
Skin with mammary gland(f)
Spinal cord
Cervical
Thoracic
Lumbar
Spleen
Testes (2)(b)
Thymus
Heart
Thyroid [with parathyroids, if present (2)(e)
Kidneys (2)
Liver (sections of two lobes)
Trachea
Lungs (including bronchi, fixed by inflation with fixative)
Urinary bladder
Uterus
Vagina
All 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 will be weighed fiom all animals at the scheduled necropsy:
Adrenals
Liver
Brain
Ovaries with oviducts
Epididymides (total and cauda)*
Spleen
Heart
Testes*
Kidneys
Thymus
*- These paired organs were weighed separately.
Paired organs were weighed together. Organ-to-body-weight ratios were calculated from animals euthanized at the scheduled necropsies.

Microscopic examination was performed on all tissues listed under macroscopic examination from all animals in the control and 10,000 ppm groups at the scheduled necropsy. The liver and thyroid were examined microscopically from all animals in the 300 and 1,000 ppm dose groups.

Other examinations:

Estrous cycles, spermatogenic evaluations- motility and viablity assessment; morphology assessment; enumeration of epididymal and testicular sperm production rate

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 of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

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

1

Dose descriptor:

NOEL

Effect level:

1 000 ppm

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

10 000 ppm

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The results of this study support the conclusion that a clear no-observed-effect level (NOEL) for oral administration of C-4000 to Crl:CD(SD) rats for 28 consecutive days was established at the dietary concentration of 1,000 ppm [approximately 77 mg/kg/day for males and 84 mg/kg/day for females]. In addition, the minimal level of response in the liver and thyroid of the rats in the 10,000 ppm group [approximately 723 mg/kg/day for males and 898 mg/kg/day for females] supports the conclusion that these liver and thyroid changes are an adaptive response, in contrast to toxicity. Therefore, oral administration of C-4000 at the dietary concentration of 10,000 ppm is the no-observed-adverse-effect level (NOAEL).

Executive summary:

The objective of the study was to evaluate the possible systemic toxic effects of C-4000 when administered orally in the diet to rats for 28 days, and to assess selected functional endpoints of the reproductive organs in males and females. The test substance, C-4000, was administered on a continuous basis in the diet for a minimum of 28 consecutive days to 3 groups (Groups 2-4) of Crl:CD(SD) rats. The dietary test substance concentrations were 300, 1,000 and 10,000 ppm active ingredient (a.i) for Groups 2-4, respectively. The diet formulations were prepared using a correction factor for purity (1.159). A concurrent control group (Group 1) received the basal diet on a comparable regimen. All groups (Groups 1-4) consisted of 10 animals/sex. Following 28 days of diet administration, all rats were euthanized by carbon dioxide anesthesia and exsanguinated. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Functional observational battery and locomotor activity data were recorded for all animals during the last week of administration (study week 3). Ophthalmic examinations were performed during study weeks -1 and 3. Clinical pathology evaluations (hematology, coagulation and serum chemistry) were performed for all animals at the scheduled necropsy (study week 4). Vaginal lavages to determine the stage of estrous were performed daily for all female animals beginning on study day 14 and continuing through the day prior to necropsy. Complete necropsies were conducted for all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from all animals. Spermatogenic endpoints (motility/viability,morphology, enumeration of epididymal and testicular sperm and sperm production rates) were evaluated for all males at the scheduled necropsy. The analytical data indicate the rats in this study received the correct dosages of C-4000 in their feed; 300 ppm [approximately 22 mg/kg/day for males and 26 mg/kg/day for females]; 1,000 ppm [approximately 77 mg/kg/day for males and 84 mg/kg/day for females]; and 10,000 ppm [approximately 723 mg/kg/day for males and 898 mg/kg/day for females]. Control diets contained no C-4000. There were no test substance-related effects on survival, clinical observations, body weights, food consumption, functional observational battery, locomotor activity, ophthalmic examinations, hematology parameters or estrous or spermatogenic evaluations. The mean serum cholesterol for the 10,000 ppm group males was above the WIL Historical Reference Range Values (version 2.6) for 9 to 12 week old male rats and was considered a treatment-related effect. Male and female rats in the 10,000 ppm group had higher absolute and relative to body liver weights, but the differences were statistically significant only on a relative basis. These weight changes were consistent with microscopic findings of minimal centrilobular hepatocellular hypertrophy in the liver. The microscopic observations of the liver and thyroid were judged to be related to treatment with C-4000 only at the high dose, but at this dose the responses were below the threshold for toxicity. The types of responses observed, the minimal level of severity, and the incidence data were indicative of an adaptive response in this species, rather than a manifestation of overt toxicity. There was minimal enlargement of centrilobular hepatocytes due to cytoplasmic hypertrophy of a few 10,000 ppm group males and females. The liver cells were enlarged due to a minimal increased amount of dense eosinophilic cytoplasm, which resulted in a more prominent appearance of the central zones of the hepatic lobules. This minimal hepatocellular hypertrophy could correlate with the minimal, but statistically significant increase in liver weights when expressed as the liver to body weight ratio and was considered to be a test substance-related effect. Minimal or mild hypertrophy and hyperplasia of the follicular epithelial cells of the thyroid was observed in animals of all groups, including controls, with a higher incidence in the high dose group. Although this thyroid change occurred in animals of other groups without hepatocellular hypertrophy, the slight increased incidence of the thyroid follicular epithelial hyperplasia and hypertrophy, particularly in the 10,000 ppm group females, may be related to the hepatocellular hypertrophy in the animals of this group. The mean absolute and relative liver weights were within the range of WIL Historical Control values and were not considered adverse. Thus, the liver and thyroid changes are considered to be an adaptive response, in contrast to toxicity. 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. The results of this study support the conclusion that a clear no-observed-effect level (NOEL) for oral administration of C-4000 to Crl:CD(SD) rats for 28 consecutive days was established at the dietary concentration of 1,000 ppm [approximately 77 mg/kg/day for males and 84 mg/kg/day for females]. In addition, the minimal level of response in the liver and thyroid of the rats in the 10,000 ppm group [approximately 723 mg/kg/day for males and 898 mg/kg/day for females] supports the conclusion that these liver and thyroid changes are an adaptive response, in contrast to toxicity. Therefore, oral administration of C-4000 at the dietary concentration of 10,000 ppm is the no-observed-adverse-effect level (NOAEL).