Registration Dossier

Toxicological information

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP study - performed according to OECD 408, adopted in 1981 - with acceptable restrictions - lacking information on neuro- and immunotoxicity

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
(no information on neurotoxicity and immunotoxicity)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical state: white powder
- Analytical purity: commercial grade
- Storage condition of test material: room temperature
- Solubility: practically insoluble in water
Specific details on test material used for the study:
- Analytical purity: 99.7%
- Physical state: solid
- Batch No.: EN 02885.32
- Storage: room temperature
- Stability is guaranteed by the sponsor for the duration of the experiment.

Test animals

Species:
rat
Strain:
other: RAI f (SPF); hybrid of RII 1/Tif x RII 2/ Tif
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Production, Ciba Geigy Ltd., Stein, Switzerland
- Age at study initiation: approx. 4 weeks
- Weight at study initiation: 64 - 96 g (males) and 63 - 86 g (females)
- Housing: specified pathogen free (SPF) standard laboratory conditions; groups of 5 in macrolon cages type 4 with standardised granulated soft wood bedding
- Diet: pelleted, certified standard diet Nafag No. 890 Tox; ad libitum
- Water: tap water; ad libitum (drinking water quality)
- Acclimation period: 8 days
Quality of diet and water was checked analytically.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10 During the acclimation period, relative humidities of 66% and 68% were reached on 2 seperate days, respectively. This deviation is considered to have no impact on the validity of the study.
- Air changes (per hr): 16 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Because no significant decrease of the test substance was found after a storage time of 28 days. Hence in the 90 day study, diet mixes were prepared 3-times during the 90-day study. Appropriate amounts of the test substance was mixed with pulverised food; about 25% water was added before pelleting (air-dried).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analytical method: Liquid chromatography
- Chemical analyses of the test substance in diet performed on a weekly basis over 13 weeks indicated following percentage recovery.
50 ppm = 93.7%
300 ppm = 93.7%
2,000 ppm = 95.4%
10,000 ppm = 98.6%
Duration of treatment / exposure:
13 weeks (plus 4 weeks recovery period without treatment)
Frequency of treatment:
daily for 13 weeks (in diet)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
3.7, 22.1, 152.7, 779.5 mg/kg bw (males) and 3.7, 22.5, 155.1 and 802.2 mg/kg bw (females) (calculated from food consumption and body weight and corrected for the % recovery as detected by chemical analysis)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
- Concentration in feed: 0, 50, 300, 2,000 and 10,000 ppm (mg/kg feed)
Basis:
nominal in diet
No. of animals per sex per dose:
treatment period: 20
recovery period: 10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: dose range is based on the result of a 28 d range finding study
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, documentation at least weekly

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily; documentation at least weekly
All animals were checked daily (twice on working days and once on weekends).

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption per cage was 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

WATER CONSUMPTION AND COMPOUND INTAKE: Yes (water consumption per cage was recorded)
- Time schedule for examinations: performed on a weekly basis during the first month, then on a monthly basis afterwards.

OPHTHALMOSCOPIC EXAMINATION: Yes (including observation of eye appearance and of periocular regions and functional examination using and ophthalmoscope)
- Time schedule for examinations: 5 days prior to treatment and towards the end (day 88); (day 116 in recovery period)
- Dose groups that were examined: control group and highest dose group (10,000 ppm)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment and recovery period
- Anaesthetic used for blood collection: yes (ether anesthesia)
- Animals fasted: yes (18 hours)
- How many animals: in all surviving animals
- Parameters checked in table No. 9 (see attachment) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment and recovery period
- Animals fasted: yes (18 hours)
- How many animals: in all surviving animals
- Parameters checked in table No. 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of treatment and recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (only feed was withheld)
- How many animals: in all surviving animals
- Parameters checked: urine volume, urine specific gravity, pH-value, protein, ketones, bilirubin, blood, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

At the end of the treatment period and at the end of the recovery period, all animals were exsanguinated unter ether anaesthesia, and subjected to detailed autoposy, in order to detect and record gross lesions as to location and type, and whenever possible number and size.
Besides the weight of the exsanguinated body, the following organs were weighed: brain, heart, liver, kidneys, adrenals, thymus, spleen and gonads.

The following organs and tissues were preserved in 10% neutral formalin: skin, mammary area, spleen, mesenteric lymph node, axillary lymph node, sternum with bone marrow, femur with joint skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland, liver, pancreas, oesophagus, stomach, small intestine, large intestine, kindney, urinary bladder prostate seminal vesicle testis, epididymis, uterus, ovary, pituitary gland, adrenal gland, thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve, orbital gland, extraorbital lacrimal gland.

From male and female control groups and all dosage groups of the test group and the recovery group samples of the organs (spleen, liver, kidney, testis, ovary, pituitary gland, adrenal gland, thyroid, brain) were taken after fixation, embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin and eosin and subjected to microscopic examination.
Statistics:
For each time point and parameter a uni-variate statistical analysis was conducted. Due to the routine manner of the analysis system, parameter free method were applied. Each treated goup was compared to the control group in respect of dispersion and displacement. In addition, a trend test was applied considering all groups. Statistical analysis is performed to draw attention to distinct values. Statistically significant difference between two values does not necessarily imply biological relevance of that deviation and is not conclusive for a treatment related effect.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related death occurred in the course of the study. No treatment-related clinical symptoms and no signs of systemic toxicity were observed during the study (see table 4 and 5; attachment).

BODY WEIGHT AND WEIGHT GAIN
The mean body weight in all treated male and female groups was similar to that of the respective controls over the study period (see table 6; attachment).

FOOD CONSUMPTION AND COMPOUND INTAKE
The mean feed consumption in all treated male and female groups was similar to that of the respective control groups (see table 7; attachment).

FOOD EFFICIENCY
The mean feed conversion in all treated male and female groups was similar to that of the respective control groups (see table 8; attachment).

WATER CONSUMPTION
The mean water consumption in all male and female treatment groups was within the normal range and was comparable to that of the respective control groups.
- mean consumption for males over 13 weeks + 1 acclimation; 225 g/animal/week in controls versus 233.6, 219.6, 230.6 and 231 g/animal/week in the 50, 300, 2,000 and 10,000 ppm dose groups respectively. No effects were seen in the recovery groups (259.5 in controls versus 279.5, 250, 259.5 and 277.5 g/animal/week in the 50, 300, 2,000 and 10,000 ppm dose groups respectively).
- mean consumption for females over 13 weeks + 1 acclimation; 198.7 g/animal/week in controls versus 194.9, 175, 213, and 179.2 g/animal/week in the 50, 300, 2,000 and 10,000 ppm dose groups respectively. No effects were seen in the recovery groups (211 in controls versus 191.5, 167.5, 216.5 and 193 g/animal/week in the 50, 300, 2,000 and 10,000 ppm dose groups respectively).

OPHTHALMOSCOPIC EXAMINATION
Eye examination and hearing test performed 5 days before the start of treatment and towards (day 88) the end of the treatment period, and towards (day 116) the end of the recovery period, revealed no evidence of a reaction to the treatment.

HAEMATOLOGY (see table 9; attachment)
No relevant differences between treated and control groups were found in the haematological investigations. At the end of the treatment period (week 14) a slightly higher number of white blood cells occurred in the females of group 4 and 5 (2,000 and 10,000 ppm). The biological relevance of this finding is in doubt since all the individual values of group 4 animals (2,000 ppm) are within the limits of the reference value for untreated rats, and only 2 animals of group 5 (10,000 ppm) showed values slightly exceeding the upper limit of the reference interval.

The statistically significant changes in the differential blood count (increased percentage of neutrophils and decreased percentage of lymphocytes) noted in the males of all treated groups was attributed to the unusually low or high values of the respective parameter in the controls.

There were some other changes achieving a level of statistical significance in their difference from controls which are attributed to spontaneous variations rather than to the treatment with the test compound, since the findings reflect the normal physiological variation of the parameters.

CLINICAL CHEMISTRY (see table 1)
No substance related changes were observed throughout the treated groups which could be related to the administration of the test article. The changes that occurred reflect the normal physiological fluctuation of the clinical chemistry parameters. The statistically significant lower values of plasma
cholesterol in the males of group 3, 4 and 5 (300, 2,000 and 10,000 ppm) is due to the relatively high value observed in the control group.

URINALYSIS
The values in all treated groups were generally unremarkable and comparable to those of the controls. At the end of the treatment period ketonuria occurred in 6 male animals of group 4 (2,000 ppm) and higher urobilinogen concentration was noted in the urine of 14 female rats s of group 4 (2,000 ppm). These findings could not be substantiated by similar changes in the high dose animals, and therefore are not considered treatment related.

ORGAN WEIGHTS (see table 2 and 3; also see attachment table 10 and 11) and HISTOPATHOLOGY
At the end of the treatment period, a statistically significant (p< 0.05) increase in mean liver weights, the liver to body ratio and/or liver to brain ratio was observed in males from group 5 (10,000 ppm) and 4 (2,000 ppm), and in females from group 3 (300 ppm) and above. The effect was not reversible as similarly increased liver weights were seen in males and females from group 4 and 5 at the end of the recovery period. The increased liver weights are concomitant with a slight to moderate hypertrophy and/or cytoplasmic vacuolization of hepatocytes in 10/10 males and 9/10 females from group 5 (10,000 ppm), 6/10 males and 9/10 females from group 4 (2,000 ppm), and 5/10 females from group 3 (300 ppm). At the end of the recovery period, 10/10 males and 7/10 females from group 5, 6/10 males and 2/10 females from group 4, and 1/10 female from group 3 showed similar findings in hepatocytes.
The statistically significant increase in relative and/or absolute mean liver weight noted in group 2 (50 ppm) at the end of the recovery period was not considered to be of experimental relevance, since it was not related to histopathological changes, and since it was not detected at the end of the treatment period.

All other morphological lesions observed in control and treated animals were only incidental in nature and not related to the administration of the test substance.

GROSS PATHOLOGY
Macroscopical examination at autopsy revealed no evidence of a reaction to the treatment, neither at the end of the treatment period nor at the end of the recovery period.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
22.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: - Nominal concentration = 300 ppm (mg/kg feed).
Dose descriptor:
LOAEL
Effect level:
153.9 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: - Corresponds to 2000 ppm (mg/kg feed). - Based on irreversible liver weight increases seen at this and higher doses that were also accompanied by irreversible microscopic changes in the liver.

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Blood chemistry and urinanalysis results after 14 and 18 weeks (recovery period)

Parameter

Dose in ppm

0

50

300

2,000

10,000

m

f

m

f

m

f

m

f

m

f

After 14 weeks (mean out of 20 animals per group)

Clinical chemistry

Alk. Phosphatase U/L

173.7

114.4

189.9

102.9

189.1

111.4

188.2

102.7

171.0

107.7

Ala. Aminotransf. (GPT) U/L

21.6

30.3

25.9

22.7

25.0*

22.8

25.3

21.4*

29.0

22.7

Asp. Aminotransf. (GOT) U/L

56.8

81.3

66.9

62.2

58.5*

61.5

61.0

77.6

57.2

61.3

G-Glutamyl transpeptidase

2.0

1.5

2.3

1.9*

1.8

1.8

2.2*

2.3*

1.7*

1.8

Sodium

142

140

141

140

142

139

140

137*

141

138

Potassium

3.92

3.51

3.77

3.49

3.71

3.19

3.89

3.28

3.78

3.32

Calcium

2.41

2.39

2.43

2.44

2.32

2.31

2.4

2.34

2.33

2.34

Phophat. inorg

1.66

1.51

1.53

1.32*

1.52

1.25

1.52

1.48

1.45

1.43

Urea mmol/L

5.8

6.7

5.5

6.6

5.8

6.9

5.6

6.8

5.4

6.9

Glucose mmol/L

8.6

7.1

8.6

7.6

8.0

6.9

8.4

7.8

7.8

7.6

Bilirubin

1.3

1.4

1.0

1.3

1.2

1.4

1.1

1.3

1.2

1.3

Cholesterol

2.1

2.1

1.9

2.4

1.7

2.1

1.8

2.5

1.8

2.0

Total protein g/L

68.3

65.8

68.0

69.2*

67.7

69.3*

67.9

68.6*

68.1

66.5

Albumin g/L

34.7

37.7

35.5

39.2

35.7

38.9*

35.1

38.2

35.2

38.1

A/G ratio

1.04

1.35

1.09

1.31

1.12

1.28

1.07

1.26

1.07

1.35

Total globulin g/L

33.6

28.1

32.5

30.1

32.0

30.4

32.8

30.4*

32.9

28.4

Urinanalysis

Specific gravity

1.033

1.033

1.036

1.033

1.035

1.029

1.030

1.029

1.032

1.034

Chemical findings – test strip pH

7

7

7

7

7

7

7

7

7

6

After 18 weeks (mean out of 10 animals per group)

Clinical chemistry

Alk. Phosphatase U/L

173.7

114.4

189.9

102.9

189.1

111.4

188.2

102.7

171.0

107.7

Ala. Aminotransf. (GPT) U/L

21.6

30.3

25.9

22.7

25.0*

22.8

25.3

21.4*

29.0

22.7

Asp. Aminotransf. (GOT) U/L

56.8

81.3

66.9

62.2

58.5*

61.5

61.0

77.6

57.2

61.3

G-Glutamyl transpeptidase

2.0

1.5

2.3

1.9*

1.8

1.8

2.2*

2.3*

1.7*

1.8

Sodium

142

140

141

140

142

139

140

137*

141

138

Potassium

3.92

3.51

3.77

3.49

3.71

3.19

3.89

3.28

3.78

3.32

Calcium

2.41

2.39

2.43

2.44

2.32

2.31

2.4

2.34

2.33

2.34

Phophat. inorg

1.66

1.51

1.53

1.32*

1.52

1.25

1.52

1.48

1.45

1.43

Urea mmol/L

5.8

6.7

5.5

6.6

5.8

6.9

5.6

6.8

5.4

6.9

Glucose mmol/L

8.6

7.1

8.6

7.6

8.0

6.9

8.4

7.8

7.8

7.6

Bilirubin

1.3

1.4

1.0

1.3

1.2

1.4

1.1

1.3

1.2

1.3

Cholesterol

2.1

2.1

1.9

2.4

1.7

2.1

1.8

2.5

1.8

2.0

Total protein g/L

68.3

65.8

68.0

69.2*

67.7

69.3*

67.9

68.6*

68.1

66.5

Albumin g/L

34.7

37.7

35.5

39.2

35.7

38.9*

35.1

38.2

35.2

38.1

A/G ratio

1.04

1.35

1.09

1.31

1.12

1.28

1.07

1.26

1.07

1.35

Total globulin g/L

33.6

28.1

32.5

30.1

32.0

30.4

32.8

30.4*

32.9

28.4

Urinanalysis

Specific gravity

1.033

1.033

1.036

1.033

1.035

1.029

1.030

1.029

1.032

1.034

Chemical findings – test strip pH

7

7

7

7

7

7

7

7

7

6

* Significant difference (p< 0.05) between control and group X

 Table 2: Liver weights after 14 weeks of treatment

 

Liver (g)

Liver to body weight (ratio)

Liver to brain weight (ratio)

 

male

female

male

female

male

female

0 ppm

14.1

8.3

3.3

3.1

615.4

375.9

50 ppm

14.563 (+ 3.2%)

8.214 (- 1.3%)

3.4 (+ 2.1%)

3.116 (- 1.0%)

641.175 (+ 4.2%)

379.571 (+ 1.0%)

300 ppm

15.32 (+ 8.5%)

10.498 (+ 26.1%)*

3.462 (+ 3.9%)

3.634 (+ 15.4%)*

663.393 (+ 7.8%)

487.076 (+ 29.6%)*

2.000 ppm

17.644 (+ 25%)*

11.153 (+ 34%)*

3.845 (+ 15.4%)*

3.816 (+ 21.2%)*

768.857 (+ 24.9%)*

503.568 (+ 34.0%)*

10.000 ppm

18.697 (+ 32%)*

10.648 (+ 27.9%)*

4.04 (+ 21.3%)*

3.809 (+ 21.0%)*

800.298 (+ 30.1)*

480.177 (+27.8%)*

Values in parenthesis = percentage change to control

* Significantly different from controls (p< 0.05)

 

Table 3: Liver weights after 18 weeks

 

Liver (g)

Liver to body weight (ratio)

Liver to brain weight (ratio)

 

male

female

male

female

male

female

0 ppm

15

7.9

3.0

2.3

634.2

354.0

50 ppm

15.527 (+ 2.3%)

8.535 (+ 7.7%)*

3.294 (+ 8%)*

3.092 (+ 8.5%)

645.382 (+ 1.8%)

389.441 (+ 9.2%)*

300 ppm

16.237 (+ 6.9%)

8.32 (+ 5.0%)

3.197 (+ 4.9%)

3.099 (+ 8.7%)

662.419 (+ 4.4%)

378.343 (+ 6.9%)

2.000 ppm

17.565 (+ 15.7%)

9.336 (+ 17.8%)*

3.411 (+ 11.9%)*

3.269 (+14.7%)

724.517 (+ 14.2%)

431.705 (+ 22%)*

10.000 ppm

20.617 (+ 35.8%)*

11.184 (+ 41.1%)*

4.121 (+ 35.2%)*

3.714 (+ 30.3%)*

854.257 (+ 34.7%)*

490.544 (+ 38.6%)*

Values in parenthesis = percentage change to control

* Significantly different from controls (p< 0.05)

Table 4 - 11 (see attachment)

Table 12: Historical controls: liver weights (g) (statistics): males and females week 13 -14

Project

 

831535 (n = 10)

864002 (n = 10)

850698 (n = 10)

860080 (n = 20)

 

 

 

mean

14.256

8.046

14.573

9.194

13.423

8.530

14.090

10.420

 

SD

1.807

0.0742

1.428

1.427

1.339

0.918

1.685

1.185

 

Min

12.322

7.162

11.967

7.051

11.497

7.295

11.190

8.498

 

Max

17.899

9.358

16.653

10.974

15.677

10.101

16.370

13.160

 

Applicant's summary and conclusion