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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Feb - 15 May 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted Sep 1998
Deviations:
not specified
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The strain of rat was selected as a series of studies with the test substance was already performed in rats of this strain and compiled data were available at the testing facility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 5 weeks
- Weight at study initiation: 132 - 173 g (females) and 173 - 216 g (males)
- Housing: 3 rats per cage were housed in plastic cages (41 x 26 x 20 cm) with hardwood chip bedding.
- Diet: Powdered diet CE-2 (CLEA Jpan, Inc., Tokyo, Japan), ad libitum
- Water: Drinking water (Ichinomiya City tap water), ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 60 ± 10
- Air changes (per hr): more than 15 times
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 Nov 1983 To: 22 Dec 1983

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Details on oral exposure:
DIET PREPARATION
The test material was incorporated at the required levels (without correction for the purity) into basal diet and mixed with a blender.
- Rate of preparation of diet (frequency): Weekly

VEHICLE
- Justification for use and choice of vehicle: Corn oil was added to all diets before mixing in order to prevent contamination with test material as dust.
- Concentration in vehicle: 2%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In order to analyze the content of the test material in the prepared diet, samples of the prepared diets were shipped to the sponsor. Analyses of stability and homogeneity of test material in the prepared diet had been performed prior to the commencement of the study. The homogeneity was satisfactory, and it was confirmed that test material in the diet was stable for 3 weeks in a freezer. The content of test material in the prepared diets was analyzed at every preparation. The prepared diet was started to use when the content values were confirmed to be within the acceptable range. Average contents of the test material were 2431, 4809, 9854 and 29431 ppm for the nominal concentrations of 2500, 5000, 10000 and 30000 ppm in the prepared diet, respectively.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
2 500 ppm
Remarks:
equivalent to 252.4 mg/kg bw/day for females and 175.1 mg/kg bw/day for males
Dose / conc.:
5 000 ppm
Remarks:
equivalent to 506.9 mg/kg bw/day for females and 344.1 mg/kg bw/day for males
Dose / conc.:
10 000 ppm
Remarks:
equivalent to 1019.3 mg/kg bw/day for females and 699.7 mg/kg bw/day for males
Dose / conc.:
30 000 ppm
Remarks:
equivalent to 3281.3 mg/kg bw/day for females and 2193.3 mg/kg bw/day for males
No. of animals per sex per dose:
24 (12 animals per group were euthanized and necropsied after 6 weeks treatment with the test substance)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary levels in this study were set by considering the results of a previous 28 days repeated dose toxicity study entitled at the same testing facility. No treatment related changes were observed in the highest dietary level of 30000 ppm group, the dietary level of 30000 ppm was selected as the highest dose level for this study, then 10000, 5000, 2500 ppm were selected as higher middle, lower middle and low dietary levels, respectively.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals were checked for general conditions and mortality
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Two-day food consumption per cage was measured weekly, and mean daily food consumption values per rat were calculated from the number of animals housed in one cage and the number of days. Test material intake was calculated from the nominal dietary levels, group means of food consumption and body weight data.

FOOD EFFICIENCY: Yes
Food efficiency was calculated according to a standard method.

WATER CONSUMPTION: Yes
Two-day water consumption per cage was measured weekly, and mean daily water consumption values per rat were calculated from the number of animals housed in one cage and the number of days.

HAEMATOLOGY: Yes, blood samples were collected via the abdominal aorta under ether anesthesia at pretreatment and after experimental week 6 or 13. EDTA-2K was used as anticoagulant. Wright-Diemsa solution was used for the determination of differential white blood cell percentages
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, overnight
- How many animals: All
- Parameters checked: red blood cell count (RBC), white blood cell count (WBC), haemoglobin (HB), haematocrit (HT), platelet count (PLT), differential white blood cell percentages and differential count of WBC (myelocytes, neutrophils (stab), neutrophils (segment), lymphocytes, monocytes, eosinophils, basophils)

CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes, overnight
- How many animals: All
- Parameters checked: Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total cholesterol, total proteins, albumin/globulin ratio (A/G), blood urea nitrogen, sodium (Na), potassium (K), Na/K ratio (Na/K), glucose and albumin

URINALYSIS: Yes
- Time schedule for collection of urine: Fresh urine samples were collected from the animals for scheduled necropsy at pretreatment and week 6 or 13, and were subjected to the urinalysis.
- Parameters checked: pH, proteins, glucose, ketones, bilirubin, occult blood and urobilinogen
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals for scheduled necropsy were euthanized by exsanguination under ether anesthesia at pretreatment and after experimental week 6 or 13, and subjected to gross pathological examination for body surface and internal organs.
- Organs weights were recorded using an electric balance (1205MP, Sartorius K.K., Tokyo, Japan) after gross pathological examination: Brain, thymus, heart, lungs, liver, spleen, kidneys, adrenals, ovaries, testes and pituitary

HISTOPATHOLOGY: Yes, after fixation in 10% buffered formalin solution, the organs and tissues were routinely trimmed, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin (H&E), and then examined under light microscopically. In addition, bone tissues were decalcified by the rapid decalcification method, and processed for histopathological slides.
- Organs checked: Heart, lymph nodes (mandibular, mesenteric), spleen, bone and bone marrow (femur), thymus, adrenals, pituitary, lungs, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), pancreas, liver, kidneys, ovaries, testes, spinal cord, brain (cerebrum, cerebellum), and any gross lesions
Statistics:
Statistical comparisons between control and test material treated groups of numerical data for body weights, hematology data, clinical chemistry data and organ weights (absolute and relative) were assessed using the Student’s t-test. The significances of intergroup differences in incidences of findings of gross- and histopathology were analyzed by the Fisher’s exact probability test. The significance of differences for each parameters were analyzed and evaluated at 5 % (p <0.05) or 1 % (p <0.01).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males: Body weights in the 5000 ppm group were significantly lower than control values at weeks 7, 9 and 10.
Females: Body weights in the 10000 ppm group were significantly lower than control values at weeks 8 and 11, but within the control range at the end of treatment in week 13.
These effects were not considered to be related to the test material treatment, since these were transitory and not dose-dependent.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Males: Decreased water consumptions were noted in the 30000 ppm group when compared to the control values from week 7 to week 13.
Females: Decreasing trend in water consumptions were observed in the 30000 ppm group when compared to the control values during the course of the study.
In addition, overall average water consumptions in both sexes of the 30000 ppm dose group were also reduced compared with the control group.

Since no adverse effects on general conditions, body weight, urinalysis or clinical chemistry were noted, the reduction of water consumption was not considered to be related to the test substance.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
A significantly decrease of haemoglobin values were observed in males of the 10000 ppm dose group after 6 weeks treatment with the test substance compared with the control group. After 13 weeks treatment with the test substance the hemoglobin values were within the range of the control animals.
Females:
In females of 5000, 10000 and 30000 ppm dose group a dose-dependent significantly reduced hemoglobin level after 6 weeks treatment with the test substance compared with the control group was determined.

However, the values were within the range of historical control data for rats of this strain according to data published by Charles River (reference: Charles River, Clinical Laboratory Parameters for Crl:CS (SD), March 2006). In addition, these changes were not considered to be treatment-related, since related parameters (RBC, WBC, HT and others) were not affected, and these alterations were reversible as the effects were not observed after 13-week treatment.
Please refer to Table 1 under "any other information on results incl. tables".

Reference: Charles River, Clinical Laboratory Parameters for Crl:CS (SD), March 2006; available online
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to control animals significant higher values of potassium (lower value of NA/K ratio) were noted in females of 10000 and 30000 ppm dose groups after 6-week treatment. Significant increased potassium levels and significant decreased Na/K ratio were observed in females of the 5000, 10000 and 30000 ppm dose group after 13 weeks treatment with the test substance compared with control group. This effect was not considered to be related to the treatment, since the values were within acceptable range based on historical control data determined at the testing facility.
A dose-dependent significant reduction of total-cholesterol values was determined in animals exposed to 5000, 10000 and 30000 ppm compared to control animals. This effect was not considered to be related to the treatment, since the values were within acceptable range based on historical control data determined at the testing facility.
Significant higher values of alkaline phosphatase in both sexes of the 30000 ppm dose group, significant higher values of blood urea nitrogen in females of all treatment groups and males of the 30000 ppm dose group, significant higher values of albumin in females of the 30000 ppm dose group were observed after 6-weeks treatment. However, these changes were not considered to be related to the treatment, since they were not observed after 13-weeks treatment, and no related changes were found after histopathological examination.

Please refer to Table 2 and 3 under "any other information on results incl. tables".
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
After 13 weeks treatment with the test substance only traces of occult blood were observed in 1/12 females of the control group and the 5000 ppm dose group, respectively, and in 1/12 males of the control group, 2500 and 10000 ppm dose group, respectively.
However, it was considered that these changes were not related to the treatment, and they were incidental, since dose-dependence was lacking, and any related renal change was not determined after histopathological examination.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
Significant lower absolute spleen weights (11%) were noted in males of the 5000 ppm dose group compared to controls. Significant higher relative kidney weights (8% and 9%, respectively) were observed in males of the 5000 and 30000 ppm dose group, respectively. In males of the 10000 ppm dose group a significant increase of the relative testis weight (11%) was determined.

Females:
Significant lower absolute thymus weights (17%), and significant higher relative brain weights (7%) and relative heart weights (9%) were noted in females of the 10000 ppm dose group compared to controls.

The increase of the relative kidney weight was considered not to be related to the test material treatment, since absolute kidney weight was comparable to control value, and any related renal change was not observed after histopathological examination. The effects on absolute organ weights observed in the study were not considered to be related to the test material treatment, since there were no changes in the relative organ weights and the effects occurred at individual doses without dose-dependence.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
Control: Single discolored spot (lung) in 7/12 males and small discolored testis in 1/12 males
2500 ppm: Single discolored spot (lung) in 3/12 males
5000 ppm: Crust cranium (skin/subcutis) in 1/12 males after 6-week treatment; single discolored spot (lung) in 1/12 males
10000 ppm: Single discolored spot (lung) in 2/12 males, small discolored testis, multiple discolored spots (lung) and discolored area (lung) each in 1/12 males
30000 ppm: Single discolored spot (lung) in 6/12 males, crust cranium (skin/subcutis) and reddened material perigenital (skin/subcutis) each in 1/12 males

Females:
Control: Single discolored spot (lung) in 4/12 females
2500 ppm: Single discolored spot (lung) in 4/12 females
5000 ppm: Single discolored spot (lung) in 2/12 females
10000 ppm: Single discolored spot (lung) in 3/12 females
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Heart, lymphocytic accumulation:
Control: 1/12 males (grade 1)
30000 ppm: 1/12 males (grade 1)

Pituitary, Cyst:
Control: 1/12 females (grade 1)
5000 ppm: 1/12 males (grade 1)
10000 ppm: 1/12 females (grade 1)

Lung, lymphocytic accumulation:
Control: 5/12 females (grade 1), 8/12 males (grade 1), 1/12 males (grade 2)
2500 ppm: 4/12 females (grade 1), 3/12 males (grade 1), 1/12 males (grade 2)
5000 ppm: 2/12 females (grade 2), 5/12 males (grade 1)
10000 ppm: 3/12 females (grade 1), 2/12 females (grade 2), 7/12 males (grade 1), 3/12 males (grade 2)
30000 ppm: 4/12 females (grade 1), 7/12 males (grade 1)

Lung, accumulation of foamy cells:
Control: 1/12 females (grade 1), 1/12 males (grade 1)
5000 ppm: 1/12 males (grade 1)
10000 ppm: 1/12 females (grade 1), 3/12 males (grade 1)

Lung, bronchopneumonia:
5000 ppm: 2/12 females (grade 1)

Lung, calcification arteria wall:
Control: 1/12 males (grade 1)
2500 ppm: 1/12 females (grade 1), 1/12 males (grade 1)
5000 ppm: 3/12 females (grade 1), 6/12 males (grade 1)
10000 ppm: 1/12 females (grade 1), 1/12 females (grade 2), 3/12 males (grade 1)
30000 ppm: 2/12 females (grade 1)

Large intestine, parasites in lumen:
Control: 1/12 females (grade 1), 3/12 males (grade 1)
2500 ppm: 3/12 females (grade 1), 3/12 males (grade 1)
5000 ppm: 1/12 females (grade 1), 3/12 males (grade 1)
10000 ppm: 2/12 females (grade 1)
30000 ppm: 1/12 females (grade 1), 3/12 males (grade 1)

Pancreas, atrophy of acinar cells:
5000 ppm: 1/12 females (grade 1)

Liver, fatty metamorphosis:
Control: 1/12 females (grade 1), 1/12 males (grade 1)
5000 ppm: 1/12 females (grade 1), 2/12 males (grade 1)

Adrenal, fatty metamorphosis:
Control: 1/12 males (grade 1)

Testis, atrophy:
Control: 1/12 males (grade 2)
1000 ppm: 1/12 males (grade 1)

Skin/subcutis, inflammation:
30000 ppm: 1/12 males (grade 1)

Preputial-clitoral gland, cyst:
30000 ppm: 1/12 males (grade 1)
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 30 000 ppm
Based on:
test mat.
Remarks:
equivalent to 3281.3 mg/kg bw/day for females and 2193.3 mg/kg bw/day for males
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects observed up to and including the highest tested dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
3 281.3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse and treatment-related effects observed up to and including the highest tested dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
2 193.3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse and treatment-related effects observed up to and including the highest tested dose level.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1: Summay of hematological data

Parameter   Control
males
1000 ppm
males
3000 ppm
males
10000 ppm
males
30000 ppm
males
Control
females
1000 ppm
females
3000 ppm
females
10000 ppm
females
30000 ppm
females
  N 12 12 12 12 12 12 12 12 12 12
Hemoglobin (g/dL) at week 7 Mean ± SD 14.5± 0.4 14.2± 0.6 14.1± 0.7 14.0± 0.4 * 14.2± 0.6 14.8± 0.7 14.7± 0.8 14.1± 0.5 ** 14.1± 0.4 ** 13.9± 1.3 *
Hemoglobin (g/dL) at week 13 Mean ± SD 16.2± 0.9 15.7± 0.7 15.7± 0.5 15.7± 0.7 15.4± 1.0 15.8± 0.9 15.4± 0.5 15.5± 0.8 15.1± 0.5 *

15.1± 0.9

* Significantly different from control group at p < 0.05

** Significantly different from control group at p < 0.01

Table 2: Summary of clinical chemistry data

Parameter   Control
males
1000 ppm
males
3000 ppm
males
10000 ppm
males
30000 ppm
males
Control
females
1000 ppm
females
3000 ppm
females
10000 ppm
females
30000 ppm
females
  N 12 12 12 12 12 12 12 12 12 12
Aspartate aminotransferase (KU)
at week 7 
Mean ± SD 127 ± 32 140 ± 36 124 ± 28 130 ± 34 141 ± 28 118± 23 125 ± 25 124 ± 22 132 ± 29 139 ± 30
Aspartate aminotransferase (KU)
at week 13 
Mean ± SD 131 ± 39 121 ± 24 133 ± 35 140 ± 35 134 ± 49 119 ± 35 110 ± 24 118 ± 21 114 ± 19 115 ± 21
Alanine aminotransferase (KU)
at week 7
Mean ± SD 26 ± 6 29 ± 7 32 ± 6 * 29 ± 6 30 ± 7 23 ± 6 27 ± 4 28 ± 4 * 29 ± 8 * 28 ± 10
Alanine aminotransferase (KU)
at week 13
Mean ± SD 25 ± 8 25 ± 4 28 ± 9 29 ± 9 26 ± 7 31 ± 14 29 ± 4 25 ± 6 26 ± 5 27 ± 6
Total cholesterol (mg/dL)
at week 7
Mean ± SD 54 ± 8 55 ± 8 52 ± 11 55 ± 9 52 ± 6 62 ± 13 62 ± 13 63 ± 9 61 ± 12 58 ± 12
Total cholesterol (mg/dL)
at week 13
Mean ± SD 73 ± 16 67 ± 20 57 ± 9 ** 53 ± 16 ** 57 ± 13 * 71 ± 8 80 ± 13 71 ± 8 75 ± 10 73 ± 17
Potassium (meq/L) at week 7 Mean ± SD 4.9 ± 0.3 5.1 ± 0.4 4.9 ± 0.3 5.2 ± 0.4 5.4 ± 0.4 ** 4.6 ± 0.2 4.7 ± 0.3 4.7 ± 0.4 4.9 ± 0.4 * 4.9 ± 0.3 *
Potassium (meq/L) at week 13 Mean ± SD 4.8 ± 0.3 4.7 ± 0.5 4.8 ± 0.4 4.9 ± 0.3 5.0 ± 0.4 4.0 ± 0.2 4.2 ± 0.3 4.3 ± 0.4 * 4.4 ± 0.5 * 4.5 ± 0.3 **

* Significantly different from control group at p < 0.05

** Significantly different from control group at p < 0.01

Table 3: Historical control data of CD(SD) rats at week 13 from 2 different examinations

  K (meq/dL) Na (meq/dL) Total cholesterol (mg/dL)
Female      
N 19 19 19
Mean 4.4 142 72
S.D. 0.3 3 14
N 12 12 12
Mean 4.4 143 78
S.D. 0.4 1 13
Male      
N 20 20 20
Mean 4.7 143 49
S.D. 0.6 1 7
N 12 12 12
Mean 4.7 144 68
S.D. 0.5 1 7

Applicant's summary and conclusion