Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Nov-12 Dec 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study with acceptable restrictions. The study was performed according to the 1981 version of OECD guideline 407; compared to the current version there was no neurobehavioural examination, no detailed clinical observations outside the cage, and few tissues/organs were preserved and examined.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
current version
Deviations:
yes
Remarks:
no neurobehavioural examination, no detailed clinical observations outside the cage, few tissues/organs were preserved and examined
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted in May 1981
Deviations:
no
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): AF-366
- Physical state: white powder
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
other: Wistar (Bor:WISW)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: F. Winkelmann, Institute for the Breeding of Laboratory Animals GmbH & Co., KG, Borchen, Germany
- Age at study initiation: approximately 4 weeks
- Weight at study initiation: 52.3-61.6 g (range, males), 50.0-62.5 g (range, females)
- Housing: the rats were housed 5 per cage/sex, in in suspended, stainless steel cages, fitted with a wire-screen bottom and front
- Diet: CIVO cereal-based, basal diet for rats and mice, ad libitum. Twice a week the feed remains in the feeders were replaced by new portions of the diets.
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 55-75
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 Nov 1988 To: 12 Dec 1988

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): fresh batches were prepared twice, on 11 Nov and 24 Nov 1988. The test diet with the highest concentration was prepared first. To obtain a homogeneous distribution of the test substance in the diet, AF-366 was thoroughly mixed with a small portion of stock diet in an electric coffee grinder, and then further diluted with stock diet to the intended concentration in a mechanical blender (Stephan cutter). The lower concentrations were obtained by diluting the top-dose diet with stock diet.
- Mixing appropriate amounts with (Type of food): CIVO cereal-based, basal diet for rats and mice
- Storage temperature of food: -20 °C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Immediately after preparation of the first batch of the diets, 5 samples of each test diet were taken at different locations in the blender and analysed by HPLC to determine the content and the homogeneous distribution of the test substance in the diet. The mean concentrations of test substance found in the test diets were close to the nominal concentrations. The analyses yielded coefficients of variation between 1 and 3 %, indicating a satisfactory homogeneity at all dietary levels, with a recorevy rate of 91-98%.
In separate experiments, partly performed prior to the start of the toxicity study, the stability of AF-366 in the basal diet was determined by analysing diet samples after storage at room temperature in open containers (4 days) or in a freezer at -20 °C in closed containers (14 and 21 days). The test substance was shown to be stable in the diet under the tested conditions.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250, 1750 and 12250 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
31, 205 and 1419 mg/kg bw/day (males); 28, 199 and 1373 mg/kg bw/day (females)
Basis:
other: calculated based on the food intake per cage per week
No. of animals per sex per dose:
5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the doses were selected based on the results (body weight and food intake) of a 5-day range-finding study (data not reported)
- Rationale for animal assignment (if not random): the weight of one female was more than 20% lower than the mean body weight on the day of study start. Therefore the rat was replaced by a reserve rat with a suitable body weight.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
- Time schedule: twice daily Monday-Friday and once daily on Saturday and Sunday

BODY WEIGHT: yes
- Time schedule for examinations: the body weight was recorded on Day 0 (prior to dosing) and weekly thereafter

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, food consumption per cage per week was measured and divided by the number of animals in a cage to reach the individual intake.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: no


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

WATER CONSUMPTION: yes
- Time schedule for examinations: water intake was measured daily per cage, during the first week of the study

OPHTHALMOSCOPIC EXAMINATION: no

HAEMATOLOGY: yes
- Time schedule for collection of blood: Day 23
- Anaesthetic used for blood collection: no
- Animals fasted: no
- How many animals: all the rats in all the groups
- Parameters examined: haemoglobin, packed cell volume, red blood cells, white blood cells, differential white blood cell count, reticulocytes, prothrombin time, thrombocytes, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: yes
- Time schedule for collection of blood: Day 28
- Animals fasted: no
- How many animals: all the rats in all the groups
- Parameters examined: alkaline phosphatase activity, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, total protein, albumin, urea, creatinine, total bilirubin, sodium, potassium, calcium, chloride, inorganic phosphate, cholesterol, triglycerides, phospholipids. In addition, on Day 25 a blood sample was drawn from the tail vein of all the rats in all the groups to measure the glucose level. The rats were deprived of water for 24 h and of feed for 16 h prior to this blood collection.

URINALYSIS: yes
- Time schedule for collection of urine: on Day 24-25
- Metabolism cages used for collection of urine: yes, during the last 16 hours of the period
- Animals fasted: yes, all the rats in all the groups were deprived of water for 24 hours and of food during the last 16 hours of this period
- Parameters examined: volume, density

NEUROBEHAVIOURAL EXAMINATION: no
Sacrifice and pathology:
GROSS PATHOLOGY: yes, the rats were examined for gross pathological changes. The adrenals, kidneys, liver and testes were weighed. Samples of the adrenals, spleen, heart, testes, kidneys, liver and all gross lesions were preserved in a neutral, aqueous, phosphate-buffered 4% solution of formaldehyde.

HISTOPATHOLOGY: yes. The tissue samples for microscopic examination were processed and embedded in paraffin wax. Sections were cut, stained with haematoxylin and eosin and then examined microscopically. Histopathological examination was carried out on the liver, kidneys, heart, adrenals, and spleen of all animals of the control group and of the high-dose group. Due to treatment-related morphological changes in the liver of the high-dose rats, microscopy of this organ was performed on liver samples of all the rats in the low-dose and mid-dose group.
Statistics:
Body weights were evaluated by one-way analysis of co-variance followed by Dunnett’s multiple comparison test. Red blood cell characteristics, total white blood cells, absolute numbers of lymphocytes and neutrophils, clinical chemistry values, volume and density of the urine, and organ weights were evaluated by one-way analysis of variance (ZiNOVA), followed by Dunnett’s multiple comparison test. Reticulocytes and differential white blood cell counts were analysed by the Mann-Whitney U-test. The histopathological changes were examined by Fisher’s exact probability test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
12250 ppm, males: convulsions
Mortality:
mortality observed, treatment-related
Description (incidence):
12250 ppm, males: convulsions
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
12250 ppm, males: decrease in mean body weight on Day 21 and 28
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):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
12250 ppm: decrease in hemoglobin and thrombocyte levels, both sexes
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1750 ppm: decreased alkaline phosphatase levels in both sexes
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
12250 ppm: increased liver weight
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
12250 ppm, males: increased Kupffer cells, hepatic mitotic activity
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There was no mortality during the study period.
2/5 males in the high-dose group had convulsions lasting around 1 minute; one on Day 23 and 24 and the other on Day 2, 15, 23 and 24. This may have been due to the relatively high doses the rats were exposed to. One of the rats displaying convulsions also had alopecia on Day 3. In 1/5 males, encrustations on the nose was noted on 10 days during the first 2 weeks. No clinical signs were observed in the females.

BODY WEIGHT AND WEIGHT GAIN
The body weight of males in the high-dose group was statistically significantly reduced (by approximately 10%) on Day 21 and 28, compared to the control group on the same days, respectively. The two rats suffering from convulsions had a lower weight gain, compared with the other rats in the group, thereby affecting the mean value (see Table 1). There was no statistically significant difference in body weight between the female groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The food consumption was comparable between the control and treatment groups. The intake of AF-366 per kg bw in successive weeks showed the normal decrease with increasing study duration for each of the three test groups. This is due to the decrease in feed intake per unit body weight with increasing age of the rats. The mean intake over the whole study period was similar in the males and females in the same dose group.

FOOD EFFICIENCY
The food efficiency was comparable between the control and treatment groups during the study period.

WATER CONSUMPTION
The water consumption was comparable between the control and treatment groups.

HAEMATOLOGY
In both male and female high-dose groups, a statistically significant decrease (approximately 25%) in the level of thrombocytes was observed, compared to the control group (see Table 2). As the reduction was notable and observed in both sexes, it is considered to be treatment-related. A statistically significant decrease was noted in the high dose group for the hemoglobin level in both sexes, and for the mean corpuscular hemoglobin in males. However, the levels are within the historical data range (Technical Bulletin, Spring 1998. Charles River Laboratories, Wilmington, USA). The mean corpuscular volume in males administered the highest dose were statistically significantly decreased. A statistically significant increase in neutrophil level (absolute and percentage) in high dose males is considered to be treatment-related, while a decrease in the percentage of lymphocytes only is a consequence of the increase in neutrophiles. Although the reticulocyte levels in high-dose females were statistically significantly increased, the control level was very low compared to the treated groups, so the toxicological significance of the result is unclear. Also, no effect on the reticulocytes was seen in the males.
Statistically significant increases in several parameters in the female low-dose group are considered to be incidental, as no effect was seen in the high-dose group or in the males.

CLINICAL CHEMISTRY
The levels of alkaline phosphatase was statistically significantly decreased in males and females in the mid- and high-dose groups in a dose-related manner (see Table 3). The effect is considered to be treatment-related, with toxicological significance only in the high-dose groups, as no additional histopathological or clinical chemistry effects were observed at the mid-dose level. Aspartate aminotransferase concentrations were statistically significantly decreased in females administered the highest dose only, and all results were within the historical data range (Technical Bulletin, Spring 1998. Charles River Laboratories, Wilmington, USA), indicating an incidental effect. A statistically significant increase in potassium in the female low-dose group is considered to be incidental, as no effect was seen in the high-dose group or in the males.

URINALYSIS
No effects were observed on the urinalysis parameters.

ORGAN WEIGHTS
The relative liver weight of males and females in the high-dose group was statistically significantly increased (see Table 4). This is considered to be treatment-related and toxicologically relevant for the males, as histopathological effects in the liver were noted in the male high-dose group only.

GROSS PATHOLOGY
No treatment-related effects were observed during necropsy and gross pathology.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the liver of 3/5 males administered the highest dose, increased mitotic activity in the hepatocytes was observed (see Table 5). An increased focal accumulation of Kupffer cells in the sinusoids was noted in 4/5 high-dose male rats. No effects were observed in female rats.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 373 other: mg/kg bw/day, calculated based on the food intake per cage per week
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
205 other: mg/kg bw/day, calculated based on the food intake per cage per week
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Overall effects; clinical signs: convulsions; haematology: thrombocytes, mean corpuscular volume; clinical chemistry: alkaline phosphatase; organ weights: liver; histopathology: liver. Intake in mg/kg bw/day is calculated from 1750 ppm.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Body weights (mean ± SD)

Day

Sex/N

Control

250 ppm

1750 ppm

12250 ppm

0

M/5

56.8 ± 1.7

56.1 ± 1.8

57.2 ± 1.6

56.8 ± 1.5

7

M/5

91.2 ± 2.4

86.6 ± 2.0

90.6 ± 3.1

86.4 ± 1.8

14

M/5

126.6 ± 3.3

122.9 ± 1.8

124.6 ± 3.9

118.5 ± 3.2

21

M/5

165.4 ± 3.4

159.1 ± 3.2

163.4 ± 4.5

148.4 ± 6.7*

28

M/5

196.8 ± 4.0

189.5 ± 4.6

191.5 ± 4.8

175.9 ± 6.9*

 

 

 

 

 

 

0

F/5

56.2 ± 1.7

58.0 ± 1.6

55.8 ± 1.8

56.1 ± 1.9

7

F/5

86.6 ± 1.7

86.3 ± 2.0

82.1 ± 2.7

80.9 ± 1.8

14

F/5

114.3 ± 4.0

112.8 ± 3.7

108.1 ± 2.6

104.5 ± 2.5

21

F/5

132.0 ± 4.4

133.0 ± 4.5

127.8 ± 3.7

122.6 ± 3.3

28

F/5

153.0 ± 6.7

149.8 ± 5.8

147.0 ± 4.4

137.6 ± 3.8

* p < 0.05, Covar + Dunnett’s tests (two-sided)

 

Table 2: Hematological parameters (mean  ±  SD)

Parameter

Sex/N

Control

250 ppm

1750 ppm

12250 ppm

Hemoglobin1(mmol/L)

M/5

8.3 ± 0.1

8.1 ± 0.1

8.1 ± 0.1

7.4 ± 0.3**

Hemoglobin1(mmol/L)

F/5

8.6 ± 0.1

8.6 ± 0.1

8.3 ± 0.2

8.1 ± 0.1*

Thrombocytes1(109/L)

M/5

1085 ± 37

1176 ± 49

1162 ± 29

916 ± 58*

Thrombocytes1(109/L)

F/5

1083 ± 42

1089 ± 43

1162 ± 27

918 ± 54*

Reticulocytes2(/1000)

M/5

24.8 ± 3.8

19.2 ± 2.0

25.6 ± 3.7

36.0 ± 7.2

Reticulocytes2(/1000)

F/5

9.6 ± 2.8

17.6 ± 2.1

13.2 ± 4.3

21.2 ± 3.2*

Mean corpuscular volume1(fL)

M/5

70.0 ± 1.0

70.2 ± 0.7

66.8 ± 1.1

59.1 ± 3.4**

Mean corpuscular volume1(fL)

F/5

68.0 ± 1.6

71.0 ± 1.3

69.9 ± 1.7

69.7 ± 1.3

Mean corpuscular haemoglobin1(fmol)

M/5

1.39 ± 0.02

1.38 ± 0.03

1.33 ± 0.01

1.10 ± 0.11**

Mean corpuscular haemoglobin1(fmol)

F/5

1.36 ± 0.03

1.42 ± 0.02

1.36 ± 0.03

1.35 ± 0.02

Neutrophils1(109/L)

M/5

0.9 ± 0.1

0.9 ± 0.2

0.9 ± 0.3

2.5 ± 0.5*

Neutrophils1(109/L)

F/5

0.7 ± 0.2

0.7 ± 0.1

0.7 ± 0.2

0.8 ± 0.2

Neutrophils2(%)

M/5

7.2 ± 1.0

7.6 ± 2.0

8.0 ± 2.5

16.6 ± 2.3**

Neutrophils2(%)

F/5

9.2 ± 2.5

7.8 ± 1.7

8.8 ± 2.7

10.2 ± 2.0

Lymphocytes1(109/L)

M/5

11.6 ± 0.4

11.8 ± 1.5

10.7 ± 1.1

11.8 ± 1.2

Lymphocytes1(109/L)

F/5

6.6 ± 0.2

8.3 ± 0.3**

6.6 ± 0.2

6.6 ± 0.4

Lymphocytes2(%)

M/5

91.4 ± 1.2

91.6 ± 2.0

91.2 ± 2.9

82.6 ± 2.2**

Lymphocytes2(%)

F/5

88.6 ± 2.2

91.4 ± 1.5

89.0 ± 2.5

88.2 ± 1.8

1* p < 0.05, ** p < 0.01, Anova + Dunnett’s tests (two-sided)

2* p < 0.05, ** p < 0.02, *** p < 0.002, Mann/Whitney U-test (two-sided)

 

Table 3: Clinical chemistry parameters (mean  ±  SD)

 

Parameter

Sex/N

Control

250 ppm

1750 ppm

12250 ppm

Alkaline phosphatase(U/L)

M/5

429.8 ± 19.2

381.0 ± 15.7

337.6 ± 14.1**

302.8 ± 22.4**

Alkaline phosphatase(U/L)

F/5

273.3 ± 20.1

272.3 ± 11.4

190.5 ± 4.3**

135.4 ± 5.9**

Aspartate aminotransferase (U/L)

M/5

70.7 ± 1.8

63.2 ± 1.8

64.6 ± 2.3

85.9 ± 12.1

Aspartate aminotransferase (U/L)

F/5

67.6 ± 1.0

65.8 ± 2.0

58.6 ± 1.9**

58.6 ± 1.5**

* p < 0.05, ** p < 0.01, Anova + Dunnett’s tests (two-sided)

Table 4: Liver weights

 

Parameter

Sex/N

Control

250 ppm

1750 ppm

12250 ppm

Relative liver weight (g/kg bw)

M/5

44.2 ± 1.0

 

44.36 ± 1.4

 

44.46 ± 1.3

51.26 ± 1.3**

Relative liver weight (g/kg bw)

F/5

42.46 ± 0.5

41.46 ± 0.6

43.56 ± 0.8

49.06 ± 1.3**

* p < 0.05, ** p < 0.01, Anova + Dunnett’s tests (two-sided)

Table 5: Histopathological results

 

Parameter

Sex/N

Control

250 ppm

1750 ppm

12250 ppm

Focal increased number of Kupffer cells

M/5

0/5

0/5

0/5

4/5*

Focal increased number of Kupffer cells

F/5

0/5

0/5

0/5

0/5

Hepatocellular mitotic increase

 - slight

- moderate

M/5

 

 

0/5

0/5

 

 

0/5

0/5

 

 

0/5

0/5

 

 

1/5

2/5

Hepatocellular mitotic increase

 - slight

- moderate

F/5

 

 

0/5

0/5

 

 

0/5

0/5

 

 

0/5

0/5

 

 

0/5

0/5

* p < 0.05, ** p < 0.01, pairwise (Fisher’s) test

Applicant's summary and conclusion