Ceraphyl 55

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

other: clear liquid

Details on test material:

- Name of test material (as cited in study report): Ceraphyl 55
- Data received: 7/01/04
- Physical state: clear liquid
- Sample preparation: Used as received
- Lot/batch No.: P1743
- Storage condition of test material: Room temperature and humidity

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ace Animals, Boyertown, PA
- Received on: 7/13/04
- Date of birth: 5/25/04
- Weight at study initiation: 164-195 grams
- Housing: The animals were housed 1/cage in suspended stainless wire bottom cages and individually identified by a uniquely numbered ear tag.
- Diet (e.g. ad libitum): Certified Purina Rodent Chow Diet #5002M was provided ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 67-72
- Humidity (%): 50-94
- Photoperiod (hrs dark / hrs light): 12 hr light/12 hr dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
Group 1-Control group (Distilled Water)
Volume: 1.0 ml/kg (Day 1)-1.15 ml/kg (Day 2-28)
Animals: 5 males - 5 Females

Group 2- 1000 mg/kg/day-High Dose
Volume: 1.15 ml/kg
Animals: 5 males - 5 Females

Group 3- 500 mg/kg/day-Mid Dose
Volume: 0.57 ml/kg
Animals: 5 males - 5 Females

Group 4- 500 mg/kg/day-Low Dose
Volume: 0.115 ml/kg
Animals: 5 males - 5 Females


VEHICLE
- Distilled Water
- Source: MB Research Laboratories
- Description: Clear liquid
- Sample preparation: Used as received
- Storage: Room temperature and humidity

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 Days

Frequency of treatment:

The test article was administrated once daily, by gavage

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 500 and 1000 mg/kg
Basis:
nominal in water

No. of animals per sex per dose:

5 females and 5 males

Control animals:

yes

Details on study design:

Five groups of five females and 5 males Wistar albino rats were dosed orally with Ceraphyl 55 using a syringe and 16 gauge call-tipped feeding needle. Doses were administrated once daily, for 28 consecutive days.
Based on the results of a preliminary screening, 1000 mg/kg/day was chosen as high dose (group 2).
The mid (group 3) and low (group 4) doses selected were 500 and 100 mg/kg/day.

Positive control:

Group 1-Control group (Distilled Water)
Volume: 1.0 ml/kg (Day 1)-1.15 ml/kg (Day 2-28)
Animals: 5 males - 5 Females

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily, all animals were observed for toxicity, pharmacological effects and twice daily for morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on the day following receipt, at the end of the first week of equilibration, before the test, weekly, at death and on the day prior to study termination for the survivors.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Measured amounts of diet were present weekly. The amount consumed was calculated each week.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On Day 28, animals were fasted overnight, and on Day 29 each animals was anesthetized with ether prior to collecting blood from aorta.
- Parameters checked: red blood cell count, haemoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration, white blood cell count, differential leukocyte count, platelet count and prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: same as haematology
- Parameters checked: magnesium, sodium, alanine aminotransferase, sorbitol aminotransferase, sorbitol dehydrogenase, blood urea, nitrogen, total bilirubin, calcium, phosphorous, aspartate aminotransferase, albumin, globulin, total cholesterol, triglycerides, chloride, potassium, alkaline phosphatase, gamma glutamyl transpeptidase, creatinine, glucose, total proteins.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: On Day 25, gross neurotoxicity and neurobehavioral changes was conducted in the surviving animals

Sacrifice and pathology:

On day 29, all animals were humanely sacrificed using either and exsanguination.
Gross necropsy was performed in each animal. Examinations included the external surfaces of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
The adrenals, kidneys, liver and testes (with epididymides, thymus, spleen, brain and heart were trimmed and weighted wet after dissection.

Statistics:

A mean and a standard deviation was calculated for all non-discrete data from clinical chemistry, haematology, organ weights, body weights, organ/body weight ratios, functional observation battery and food consumption.
An Analysis of Variance (ANOVA) was performed on absolute organ weights, body weights, organ/body weight ratios, clinical chemistry and haematology.
The results were calculated based on the relationship between the dose levels and incidence or severity of response. The No Observed Effect Level (NOEL) was estimated.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

sodium, potassium, globulin, chloride, alanine aminotransferase (SGPT) and glucose were significantly different compared to the control group

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean adrenal weights of animals dosed at 500 mg/kg/day were greater than the controls. The organ/body weight relationship of heart, liver and kidney was significant different from the control group.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

MORTALITY:
There was no mortality due to the administration of the test material. One animal in group 2 was found dead on Day 22. Necropsy showed that the death was due to an inadvertent gavage accident.

CLINICAL SIGNS:
Group 1: Instances of localised alopecia were observed in only 2 animals.
Group 2: Instances of emaciation, wetness of the anogenital area, few faeces and a bent tail were noted in one female survivor.
Group 3: Red and swollen ears were noted in one female from Day 7 to Day 10. No abnormal signs were noticed in this animal.
Group 4: Instances of localised alopecia were noted in one animal from Day 17 to Day 26.

BODY WEIGHT AND WEIGHT GAIN:
No significant differences were recorded between control body weights and mean body weight treated with the test material. Only at Day 8 the mean of body weights of group 3 female animals was significantly less than the control.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
No significant differences in mean food consumption were recorded.

HAEMATOLOGY:
The mean prothrombin time in male animals in group 2 was significantly greater than the male controls. There were no other significant differences between any groups at any dose.

CLINICAL CHEMISTRY:
The parameters that resulted significantly different from the control group are:
Sodium: Gr4>Gr1 (Male)
Potassium: Gr4>Gr1 (Male)
Globulin: Gr2Chloride: Gr2Glucose: Gr2ALT (SGPT): Gr2>Gr1 (Female)

NEUROBEHAVIOUR:
No significant differences between groups in the orientation, sensory responsiveness posture, locomotion/patterned movements and integrated movement portion of the FOB.

ORGAN WEIGHTS:
The mean adrenal weight of group 3 females were significantly greater than the control group> No other significant different were recorded.
No significant differences were recorded for the organ/body weight ratios except:
Liver: Gr2>Gr1 (Male)
Adrenals: Gr3>Gr1 (Female)
Kidneys: Gr2>Gr1 (Female) and Gr3>Gr1 (Female)
Liver: Gr2>Gr1 (Female)
Heart: Gr2>Gr1 (Female)

HISTOPATHOLOGY:
No treatment-related microscopic changes were observed.

Effect levels

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: No treatment-related changes in male and females rats were observed

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) of the test material Ceraphyl 55 considered to be greater than 1000 mg/kg/bw.

Executive summary:

The sub-acute oral toxicity of the test material Ceraphyl 55 was investigated in 20 male and 20 females Wistar albino rats, divided in four groups. Group 1 was dosed with the vehicle (distillated water) only. Group 2, 3 and 4 were treated with Ceraphyl 55 at dose levels of 1000, 500, 100 mg/kg. Doses were administrated using a syringe and 16 gauge ball-tipped feeding middle. The highest dose was selected based on the results on a pre-screening.

All animals were observed for toxicity, pharmacological effects and twice daily for morbidity and mortality. Body weights were recorded on the day following receipt, at the end of the first week of equilibration, before the test, weekly, at death and on the day prior to study termination for the survivors. Food consumption was calculated each week. In addition haematology and clinical chemistry analysis were performed. Neurobehavioral examinations and gross necropsy was performed in each animal.

No mortalities were observed during the 28-Day administration period. One animal in Group 2 was found dead on Day 22. Necropsy showed that the death was due to an inadvertent gavage accident. Instances of abnormal physical signs of surviving animals were minimal. Significant differences were recorded between controls and animals dosed with Ceraphyl 55 in some clinical parameters. Furthermore, the mean adrenal weights of Group 3 (females) were significantly greater than the control group. Necropsy, histopathological examination and microscopic evaluation revealed no-treatment related changes in female and male rats dosed at 1000 mg/kg/day. Based on these results the NOAEL for the 28-Day Repeated dose toxicity is considered to be greater 1000 mg/kg/day.