Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 27, 1990-December 27, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Toxicological Principles for the Safety Assessment of Direct Food Aditives and Color Additives used in Food (FDA, USA, 1982)
Deviations:
yes
Remarks:
Stability testing was performed on feed mixes containing 1 and 5% of test substane after 18 weeks, rather than feed containing 0.1 and 10% of feed after 10 weeks storage in refrigeration.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: Mixed with basal diet feed.

Test animals

Species:
rat
Strain:
Fischer 344/DuCrj
Details on species / strain selection:
Strain chosen as it has been used extensively in long-term toxicity study, and therefore sufficient background data is available on it.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5 weeks
- Weight at study initiation: 73-88 g males, 74-89 g females
- Fasting period before study: No
- Housing: 2 animals in polycarbonate cages with hardwood chip bedding
- Diet: CRF-1, Oriental Yeast Co., radition-sterilized, ad libitum
- Water: filtered, UV sterilized tap water ad libitum
- Acclimation period: 5-8 days

DETAILS OF FOOD AND WATER QUALITY: Food was analyzed by a third party to ensure contaminants were below acceptable levels. Drinking water is tested biannually.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 40-70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: January 14, 1991 To: April 26, 1991

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
Since the test substance will be taken orally during actual use, it was decided to perform an oral toxicity study in feed.
Vehicle:
other: feed
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): radiation sterilized powdered feed for rats and mice


VEHICLE
- Justification for use and choice of vehicle (if other than water): appropriate feed for species selected
- Concentration in vehicle: 1, 3, and 5%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken from the top, middle, and bottom of the first batch of feed prepared. All lots of feed mix were analyzed. The stability of the test substance with checked after 18 weeks at 5-10 °C storage for the 1 and 5% mixtures. Analysis was done using gas chromatography.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Animals were given feed containing the test substance ad libitum throughout the study period.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
636 mg/kg bw/day (nominal)
Remarks:
1% of diet male
Dose / conc.:
666 mg/kg bw/day (nominal)
Remarks:
1% of diet female
Dose / conc.:
1 900 mg/kg bw/day (nominal)
Remarks:
3% of diet male
Dose / conc.:
1 950 mg/kg bw/day (nominal)
Remarks:
3% of diet female
Dose / conc.:
3 240 mg/kg bw/day (nominal)
Remarks:
5% of diet male
Dose / conc.:
3 430 mg/kg bw/day (nominal)
Remarks:
5% of diet female
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: 5% was the maximum dose recommended in the guideline.
- Rationale for animal assignment (if not random): random sampling based on body weight stratification
- Section schedule rationale (if not random):

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: survival, clinical signs, appearance, behaviour

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 3-1 days before study start, and 5-2 days before end of study
- Dose groups that were examined: control, high dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to sacrifice at end of study
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: erythrocyte count, leucocyte count, platelet count, hemoglobin concentration, hematocrit, differential leucocyte count, reticulocyte count, prothrombin time, activated partial thromboplastin time, mean corpuscular volume, and mean corpuscular hemoglobin

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to sacrifice
- Animals fasted: Yes
- How many animals: all
- Parameters checked: total protein, glucose, triglycerides, total cholesterol, phospholipids, free fatty acids, urea nitrogen, creatinine, calcium, inorganic phosphorous, GOT, GPT, gamma-GTP, ALP, albumin, A/G ratio, sodium, potassium, and chlorine

URINALYSIS: Yes
- Time schedule for collection of urine: 4-3 days before end of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH, occult blood, protein, glucose, ketone bodies, bilirubin, urobilirubin

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
General examination, weights of liver, kidneys, adrenals, testes, ovaries, brain, heart, lung, and spleen

HISTOPATHOLOGY: Yes
brain, pituitary, thyroid/parathyroids, thymus, trachea, lungs, heart, aorta, salivary glands, liver, spleen, adrenals, pancreas, testes, epididymides, prostate gland, seminal vesicle, ovaries, uterus, vagina, skin, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, bladder, lymph nodes, mammary gland, muscle, sciatic nerve, femur, sternum, eyes, spinal cord, any other organs with macroscopic changes
Statistics:
Bartlett's equal variance, ANOVA for homogenous variance, Dunnett's method was used to compare mean values between groups with the same number of animals, Scheffe's method was used to compare mean values between groups with different numbers of animals, heterogeneous variance was analyzed using the Kruskal-Wallis H-test, Data from the urinalysis was analyzed using the Armitage chi-squared test

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Bilateral alopecia of the forelimbs was noted in one female in the low dose group at 10 weeks. As this was not seen in any other group, it was not considered to be related to treatment.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Suppression of weight gain was noted in the medium dose male group in the early to mid-part of the study period. As this effect was not dose dependent, it was not considered treatment related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Males in the high dose group showed increased food consumption. The increase was not consistent, and the difference was slight. When the weight of test substance was subtracted from the total food weight, the actual consumption in this group was identical.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
A decrease in food efficiency was observed in some males animals, but this effect was short-term.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Females in the high dose group showed prolonger activated partial thromboplastin times. As the value was still within the normal range of background data, this effect was concluded to be non-treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A significant increase in albumin, and elevation in GPT in the medium and high dose male groups. The high dose males also showed a significant increase in potassium. The increase in albumin and potassium was not considered treatment related. The female high dose group also showed significant elevation of GPT. The increase in GPT values was dose-dependent, but within the range of the control group. Since no changes were seen in the livers, this effect, though treatment related, was considered to be of minor importance. A longer term study would be needed to determine the full significance of this increase. Males in the medium dose group and females in the low dose group showed decreased levels of sodium. As this decrease was not dose-dependent, it was not considered treatment related.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
A decrease of ketone bodies was observed in males in the high dose group. However, this effect is not considered to be toxicologically significant.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
High and medium dose males showed a significant decrease in absolute kidney weights. A significant decrease in relative kidney weights was seen in all male treatment groups. A decrease in relative liver weight was seen in the high dose male group. These differences were 5% or less, and no other changes relating to the decrease were observed. Thus, these effects were not attributed to treatment. A significant decrease in absolute adrenal weight was seen in the medium dose male group. This decrease was attributed to the decrease in average body weight of the treatment groups, and so the effect was not considered significant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One female in the medium dose group showed a unilateral ovarian cyst. Ovarian bursa cysts were also in one female in the low dose group, and two in the medium dose group. Hydrometra was seen in one female in the control, medium, and high dose groups. Partial alopecia was seen in one female in the low dose groups. As none of theses changes was dose-related, they were not considered to be treatment related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Hyaline droplets in the epithelium of the renal uriniferous tubules were frequently found in the control and high dose male. Since there was no significant difference between the groups, this change was not considered to be treatment related. Other changes noted in various groups were focal myocardial degeneration, dilation of the tracheal glands, calcinosis of the reanal cortico-medullary junction, and regeneration fo the uriniferous tubules. These changes were not dose dependent, and therefore not considered to be treatment related. Changes noted in less than 5 males or females, and therefore not considered to be treatment related, were increased extramedullary hematopoiesis in the spleen, lymphocytic infiltration of the tracheal submucosa, cellular infiltration in Glisson's sheath and focal necrosis in the liver, interstitial ductal proliferation and lymphocytic infiltration in the pancreas, lymphocytic infiltration of the hyaline casts and interstitial tissue in the kidney, interstitial lymphocytic infiltration of the thyroid gland, and interstitial lymphocytic infiltration of the Harderian glands. Cysts formed by the dilation of the lymphatic vessels of the ovaries, hydrometra, and hair loss were also observed. These did not occur often enough to be considered treatment related.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Focal osseous metaplasia of the alveolar wall was seen in less than 5 males or females.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
3 240 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Remarks on result:
other: No adverse effects observed up to the highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
3 430 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Remarks on result:
other: No adverse effects observed up to the highest dose tested.

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
3 240 mg/kg bw/day (nominal)
System:
other: blood
Organ:
blood

Applicant's summary and conclusion

Conclusions:
The NOAEL for male rats was 3240 mg/kg bw/day (5% of diet), and the NOAEL for female rats was 3430 mg/kg bw/day (5% of diet).
Executive summary:

The oral toxicity of the test substance was determined in a 13-week feed study. The substance was added to the diet at 0, 1, 3, and 5%. Groups of 20 male and 20 female rats were tested at each dose level. After 13 weeks, the animals were sacrificed and necropsied. A slight mean increase in GPT activity of the medium and high dose males, and high dose females was noted. As all the observed values were within the control range, a longer term test would be needed to determine if this was treatment related. As there were no definitive treatment related effects, the NOAEL for both males and females was the highest dose, 5% of feed. For males, this was the equivalent of 3240 mg/kg bw/day, and for females 3430 mg/kg bw/day.