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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): FAT 80´023
- Physical state: white powder
- Analytical purity: 99%
- Lot/batch No.: 5.2.0211.0

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York
- Age at study initiation: 37 days
- Weight at study initiation: males 155.5 to 161.1 g, females 127.6 to 129.2 g
- Fasting period before study: no
- Housing: individually in wire-bottom, suspended cages
- Diet (e.g. ad libitum): certified Purina Rodent Chow® No. 5002, ad libitum
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: at least 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hrs/12hrs

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Batches of test compound blended into the diet at different scheduled concentrations were prepared once a week during the study.
The prepared diets were used within 1 week of preparation and were stored at room temperature.
The homogenous blends of the test compound/diet mixtures were given to the experimental groups ad libitum and in excess; the control groups received the powdered diet without test compound.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Serial sacrifice groups were fed with the test compound in the diet for 13, 26, and 78 weeks;
The 1-year interim sacrifice groups were fed the test compound in the diet for 52 consecutive weeks;
The 2-year carcinogenicity groups were fed the test compound in the diet for 104 consecutive weeks.
Frequency of treatment:
daily, 7 days a week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 300, 1000, 3000 ppm, and 6000 ppm (52 weeks only)
Basis:
nominal in diet
No. of animals per sex per dose:
Each of the serial groups (13, 26, and 78 weeks): 15 animals/sex/group
The 1-year interim sacrifice groups (52 weeks): 20 animals/sex/group
The 2-year carcinogenicity groups (104 weeks): 60 animals/sex/group
The toxic level dose group (6000 ppm, 52 weeks): 20 animals/sex
The baseline pre-dose group (0 ppm): 10 animals/sex
Control animals:
yes, concurrent no treatment
Details on study design:
The 0, 300, 1000 and 3000 ppm dose levels:
The 3000 ppm dose level corresponded to the maximum tolerated dose (MTD)
For each of these dose levels, three groups of animals (both sexes) were considered:
- A serial group with serial scheduled sacrifices after 13, 26, and 78 weeks,
- A one-year interim group, with scheduled sacrifice after 52 weeks,
- A two-year carcinogenicity group, with scheduled sacrifice after 104 weeks.

The 6000 ppm dose level was designated as the toxic dose level group with scheduled sacrifice after 52 weeks.
The baseline pre-dose group served for optention of baseline data referring to haematology and clinical chemistry.

Examinations

Observations and examinations performed and frequency:
GROSS PHYSICAL CHECK
At initiation of the study and monthly thereafter, all animals were examined for gross physical defects. Palpations for tissue masses were conducted monthly during the first 12 months of the study and every 2 weeks thereafter.

OPHTHALMOSCOPIC EXAMINATION
The eyes of each rat were examined during the acclimation period and during weeks 52 and 104,

MORTALITY AND CLINICAL SYMPTOMS
Each animal was monitored twice daily for mortality as well as toxicological and pharmacological effects. On weekends and holidays, observations were made only once daily.

BODY WEIGHT
The body weight was determined weekly for each animal during the first 12 weeks of the dosing period, and once every 4 weeks thereafter. Body weights were also recorded prior to dosing on the first day of the study.

FOOD CONSUMPTION
Food consumption was determined weekly for each animal during the first 12 weeks of the dosing period, and once every 4 weeks thereafter.

HAEMATOLOGY AND CLINICAL CHEMISTRY
Blood was collected following an overnight fast and was used for clinical chemistry and following EDTA-treatment, for haematology.
Baseline data were obtained from 10 rats/sex pre-dose.
Twenty rats/sex/group were randomly chosen from the chronic study groups and
permanently assigned as clinical test animals.
Twenty rats/sex in the group treated with 6000 ppm were also used for clinical tests. Clinical laboratory tests were conducted during study weeks 13, 26, 52, 78 and 104.
The following haematological parameters were examined in 20 animals/sex/group: hemoglobin, haematocrit, erythrocyte count, total and differential leukocyte counts, reticulocyte count (Evaluated in the control and 6000 ppm dose groups only), clotting time (not examined in animals assigned to baseline pre-dose group).
The following clinical chemical parameters were examined in 10 animals/sex/group: total protein, blood urea nitrogen (BUN), albumin, total bilirubin, albumin/globulin ratio, serum alanine aminotransferase (SGPT), glucose serum aspartic aminotransferase (SGOT), total cholesterol, gamma glutamyl transpeptidase (y-GT), triglycerides, serum alkaline phosphatase (SAP).

URINALYSIS
Following parameters were evaluated in the 10 rats/sex/group selected for clinical chemistry: specific gravity, glucose, pH, bilirubin, ketones, protein.
Sacrifice and pathology:
NECROPSY
Necropsy was performed on each animal which survived the scheduled experimental period, died, or was sacrificed moribund.

FINAL BODY WEIGHT
The animals were weighed a last time prior sacrifice.

ORGAN WEIGHT
In the serial groups sacrificed after 13, 26 and 78 weeks only the livers were weighed.
For all animals sacrificed after 52 and 104 weeks, adrenals, heart, liver, kidneys, gonads, spleen and brain were weighed. Relative organ weights were calculated as a percent of the final body weight.

GROSS PATHOLOGY
The animals were examined for macroscopical abnormalities.

ORGAN /TISSUE SAMPLING
Following organs and tissues were fixed with neutral buffered 10% formaldehyde for the purpose of (histo)pathological examination:
all gross lesions, all tissue masses, brain, pituitary, eyes and optic nerves, salivary glands, thyroids and parathyroids, trachea, esophagus, stomach, duodenum, spinal cord, jejunum, ileum, colon, thymus, heart, aorta, lungs, liver, pancreas, kidneys, adrenals, spleen, sciatic nerve, rectum, cecum, lymph nodes (mesenteric), urinary bladder, gonads, prostate, epididymides, uterus (horn and cervix), bone marrow (sternum), skeletal muscle, skin, mammary gland (both sexes), vertebra/femur.

HISTOPATHOLOGY
The livers of rats from all groups and the pancreas of rats in the interim sacrifice groups were examined histologically.
All tissues from the control and 6000 ppm dose animals assigned to the 1-year interim sacrifice group were evaluated microscopically as were tissues from any animal which was killed or which died prior to its scheduled necropsy date.
Microscopic examinations were also performed on all tissues from all rats of the control and 3000 ppm groups that were sacrificed after 2 years.
Microscopic examinations were also performed on the livers, kidneys, and lungs from all rats of the 300 and 1000 ppm groups that were sacrificed after 2 years.
All tissue masses and lesions were examined histopathologically, irrespective of the animals dose group or scheduled sacrifice date.
Other examinations:
A minimum of 2 mL of heparinized blood and a minimum of 0.5 g kidney, 3 g liver, 0.5 g spleen, 0.5 g heart, 1 g brain, 1 g skeletal muscle, and 1 g retroperitoneal fat were collected from all rats at the time of scheduled necropsy at weeks 13, 26, 52, 78; and from 50% of the surviving rats at week 104; the samples served for analyzes of the levels of test compound and its metabolites.
Statistics:
For body weight, food Consumption and organ weight, Bartlett's test for homogeneity of variance was used. If the Bartlett's test was not significant at p < 0.05, then a statistical comparison of treated group means to control group means was performed using Dunnett's t test. If Bartlett's test for homogeneity of variance was significant, then Behren's t test with Cochran's approximation was applied. Appropriate transformations (e.g., logrithmic, or square root) were applied to a predetermined set of parameters which were not normally distributed.
For the clinical parameters, analyses designed mainly to test each parameter for possible trends existing between treatment groups that comprise different doses of the same compound and a zero dose control were applied. If a significant trend is found, the test procedure is applied again to the remaining treatment groups, excluding the highest dose group, and so on, in order to examine the significance of comparisons of dose groups against the control.
Survival distribution for each group and each sex were determined using Kaplan-Meier estimates. Two-sided nonparametric tests (Gehan- Wilcoxon test and Mantel-Cox logrank test) were performed to test for differences between the survival curves of the treatment groups. If significant differences were found, multiple comparisons were then performed to compare each treated group versus the control.

Results and discussion

Results of examinations

Details on results:
No significant compound-related effects on mortality occurred during the study, mortality for all groups ranged between 40 and 50%.
Clinical signs were observed during the course of the study, which either were common to both, treated and control animals (e.g., alopecia, chromodacryorrhea, sore foot, and sore tail) or were incidental.
Ophthalmoscopic findings were incidental and/or age related and unrelated to treatment.
Significant reductions in average body weight relative to control means were noted in groups treated with 6000 ppm (both sexes) and 3000 ppm (females only); no such reductions were seen at 1000 and 300 ppm.The percent body weight difference, relative to the control group was greater for females (up to 10%) than for males.
Increased food consumption was reported for the 6000 and 3000 ppm males but not in the females.
In the treated groups, changes in haematology and clinical chemistry were slight and almost transient, but statistically significant (p ≤ 0.05) when compared to controls at various time points. The hematological alterations were generally slight and transient in nature and were considered biologically significant only at doses > 3000 ppm; although statistical differences also occurred at lower doses. The notable changes included decreased mean hemoglobin, RBC count, hematocrit, monocytes and reticulocytes (reticulocytes decreased at 6000 ppm only); and increased clotting time. Particularly the decreases in monocytes and white blood cells in females and increased clotting time in males were observed at test ending, i.e., after 104 weeks.
Slight changes in MCV, MCHB, and MCHC were observed, which were without biological significance.
Referring to clinical chemistry, statistically significant (p ≤ 0.05) differences between treated groups and controls were noted at various time periods. In general, the changes were slight and transient and were considered biologically significant only at doses ≥ 3000 ppm; although statistical differences also occurred at lower doses. The changes included increased SGPT, SGOT, blood urea nitrogen (BUN), and albumin/globulin ratio, and decreased total bilirubin, triglycerides, glucose, total serum protein, albumin. The changes in protein, glucose, bilirubin, triglyceride, and BUN were observed in the first 52 weeks of the study and had disappeared by 78-104 weeks.
Urinalysis revealed several slight but statistically significant changes at various test periods predominately at doses ≥ 3000 ppm. Urinary specific gravity and pH values were either increased or decreased, with respect to the control levels, at weeks 13 and 104; whereas, urinary proteins were reduced at doses ≥ 3000 ppm
At necropsy, no treatment-related gross lesions were noted in rats from any group throughout the study.
Statistically significant changes in average organ weights were noted in treated rats. In fact, absolute adrenal weights were increased in mid-dose males; absolute brain weights were decreased in high-dose males; absolute and relative ovary weights were increased in high-dose females; absolute and relative spleen weights were decreased in mid-dose females, and relative spleen weights were decreased in high-dose females at 104 weeks. The organ weight changes were generally associated with decreased mean body weight at doses ≥ 3000 ppm. However, at week 52, males in the 3000 ppm group had decreased mean relative liver weight in comparison to the control group.
Microscopic evaluations revealed enlarged centrilobular hepatocytes containing hyaline-appearing cytoplasmic “inclusions" in the 3000 and 6000 ppm males at their scheduled sacrifice interval of 13 and 52 weeks, respectively. Hepatocellular hypertrophy was also present but without being statistically significant in 2 out of 5 male rats in the 3000 ppm group at 78 weeks. These lesions were not observed in rats at 104 weeks. No other histological lesions, were observed that were considered to be treatment-related. With respect to neoplastic changes and tumor incidence, there were no differences between treated and control groups at 104 weeks.
With respect to neoplastic changes and tumor incidence, there were no differences between treated and control groups at 104 weeks.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Sex:
male/female
Basis for effect level:
other: (corresponding to ca. 33.5 and 34 mg/kg bw/day for males and females, respectively)
Dose descriptor:
LOAEL
Effect level:
3 000 ppm
Sex:
male/female
Basis for effect level:
other: (corresponding to ca. 107 and 113 mg/kg bw/day for males and females, respectively); based on reduced WBC counts in females and increased clotting time/decreased monocyte count in males.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Analytical monitoring:

Routine chemical analyses of the test substance/feed admixtures during the study indicated that the test substance was homogeneously distributed in the diet mixes offered to the rats. The test substance in the dietary admixtures was stable during theperiod of time from preparation to use. Furthermore, the actual concentration of FAT 80'023 in the feed was between 94% and 105.2% of the target concentrations for all dose groups during the 2-year study period, and no test substance was detected in the control feed on any assessment occasion.

Mean daily test item intake:

concentration of test item in feed (ppm)

Mean daily intake of test item (mg/kg bw/day)

Males

Females

0

0

0

300

9.31 +/- 1.89

10.65 +/- 4.01

1000

33.57 +/- 9.07

33.98 +/- 11.76

3000

107.22 +/- 26.53

113.90 +/- 25.47

Clinical chemistry, haematology and urinalysis

Parameter changed

Controls

300 ppm

1000 ppm

3000 ppm

6000 ppm

Dose-related

 

Clotting time

13 weeks

+

26 weeks

+

52 weeks

78 weeks

/

/

104 weeks

/

/

+

WBC count

104 weeks

/

/

+

Monocytes

13 weeks

26 weeks

104 weeks

/

/

+

Bilirubin

13 weeks

+

+

26 weeks

52 weeks

+

+

104 weeks

/

/

Triglycerides

13 weeks

+

26 weeks

+

+

52 weeks

+

104 weeks

/

/

ALT (GPT)

13 weeks

+

26 weeks

+

52 weeks

+

78 weeks

/

/

+

104 weeks

/

/

AST (GOT)

13 weeks

+

+

52 weeks

+

78 weeks

/

/

+

104 weeks

/

/

BUN

13 weeks

+

26 weeks

+

52 weeks

78 weeks

/

/

 

104 weeks

/

/

 

Urine, specific gravity

104 weeks

/

/

+

Summary of main findings 

Parameter changed

Controls

300 ppm

1000 ppm

3000 ppm

6000 ppm

Dose-related

 

52-wk mortality [%]

0

0

0

0

0

0

0

10

5

0

104-wk mortality [%]

63.3

66.7

70.0

68.3

53.3

65.0

63.3

70.0

/

/

52-wk bw

+

+

104-wk bw

/

/

Liver weight, relative

52 weeks

Liver, hepatocytes with inclusion bodies

13 weeks

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

4/5

0/5

+

52 weeks

0/20

0/20

0/10

0/10

0/10

0/10

0/10

0/10

4/20

0/20

+

Liver, hepatocellular hypertrophy

13 weeks

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

5/5

0/5

+

52 weeks

0/20

0/20

0/10

0/10

0/10

0/10

0/10

0/10

12/20

0/20

+

Applicant's summary and conclusion