Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
30 Nov Nov 1992 - 05 Mar 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-compliant, comparable to guideline study with acceptable restrictions. No functional observations performed; most but not all haematological/clinical chemistry, organ weight and histopathological examinations performed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no functional observations performed; most but not all haematological/clinical chemistry, organ weight and histopathological examinations performed
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): Dimer Acid
- Physical state: Yellow liquid
- Substance type: UVCB
- Analytical purity: no data
- Composition of test material, percentage of components: dimer acid, 73%; trimer acid, 21%
- Source: Unichema, Gouda; Holland

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 114.5-155.5 g (male); 104.6-142.0 g (female)
- Housing: 5 animals/cage
- Diet: ESL modified AIN-76A (MODAIN) purified diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 30 Nov 1992 To: 01-05 Mar 1993

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): experimental diets were initially prepared weekly from 26 Nov to 10 Dec 1992. After obtaining data confirming test material stability in diet for 14 days, diets were prepared every two weeks from 17 Dec 1992 to the end of the study.
- Mixing appropriate amounts with (Type of food): ESL modified AIN-76A (MODAIN) purified diet
- Storage temperature of food: diets in sealed bags were UV-sterilised for a minimum of 10 h at ambient temperature before entry in the SPF unit, where they were stored at approx. 4 °C prior to use.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
-Stability and homogeneity in diet
The stability of the test substance at concentrations of 0.1%, 1% and 5% in diet was determined over periods of 7 and 14 days when stored at 4°C or in an animal room.
The diets were analysed to confirm that the test substance was distributed homogeneously in the diet. After mixing the diets, five samples were taken for analysis from the top, the middle and bottom centre, and left and right centre of the mixing bowl.
Diet samples were extracted into propan-2-ol and centrifuged to remove particulate matter. An aliquot was concentrated to dryness, then redissolved and analysed by HPLC on a 5µ Lichrosorb Diol column, detection by a light scattering detector. Quantitation was achieved by comparison of peak areas with external standards of the test substance.
Separation on the HPLC system was based on the interaction of the carboxylic acid of the test substance with free hydroxyl groups at the surface of the diol phase. Thus, interaction increased with the number of carboxylic acid groups. The test substance contains mixtures of mono, di and polyacids. In the assay preparation based on two peaks, di-acid denoted dimer and tri or greater (poly) acid denoted trimer.
The test substance was shown to be stable in diet over 14 days. The results for 0.1% (w/w) test substance in diet showed more variation that would normally be accepted, but the level was very close to the limits of detection of the analytical method.
Using the methods described, the test substance was shown to be mixed homogeneously in the diet at a concentration of 0.1%, 1% and 5% (w/w).

-Confirmation of achieved concentration
Diets containing 5%, 1% and 0.1% test substance prepared on 26 Nov 1992, 07 Jan 1993 and 18 Feb 1993 were analysed for the achieved concentration of the test substance after first passing the UV lock. The methodology was the same as that used for the determination of stability.
Analysis of the diets prepared on Week 1 and 13 confirmed the nominal concentration had been achieved within the expected experimental error of the analytical method. The results for 0.1% (w/w) test substance in diet showed more variation that would normally be accepted, but the level was very close to the limits of detection of the analytical method.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily, 7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.1, 1, 5% (w/w)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
74.1, 740.9, 3591.2 mg/kg bw/day (males)
Basis:
other: as calculated from the reported mean body weight and food intake data
Remarks:
Doses / Concentrations:
90.5, 854.9, 4085.5 mg/kg bw/day (females)
Basis:
other: as calculated from the reported mean body weight and food intake data
No. of animals per sex per dose:
20
Control animals:
yes, plain diet

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once on Saturdays and Sundays)
- Cage side observations included: signs of ill-health or reaction to treatment

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of the study and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No. For each cage of five animals, food intake values were determined twice-weekly and weekly values were calculated.
- 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: For each cage of five animals, water consumption values were determined twice-weekly and weekly values were calculated.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the week prior to study start and end, respectively
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the test period
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: No data
- How many animals: all
- Parameters examined: red blood cell count, platelets, haemoglobin, total white blood cells, differential white cell count (lymphocytes, neutrophils, monocytes, eosinophils, basophils, large unstained cells), mean red cell volume (derived from the red cell volume histogram), haematocrit, mean red cell haemoglobin, mean red cell haemoglobin concentration, reticulocytes, prothrombin time (PT), activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the test period
- Animals fasted: No data
- How many animals: all
- Parameters examined: sodium, potassium, calcium, magnesium, chloride, inorganic phosphate, creatinine, urea, glucose, triglyceride, total cholesterol, total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), hydroxybutyrate dehydrogenase (HBD), alkaline phosphatase (AP), pseudocholinesterase, creatine kinase, 5’-nucleotidase, gamma-glutamyltransferase, total protein, albumin, alpha1-globulin, alpha2-globulin, β-globulin, gamma-globulin, albumin:globulin ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals which survived to the end of the study received a detailed necropsy. All macroscopic abnormalities were recorded and a score was allocated as an assessment of the level of intra-abdominal fat deposition.
Brain, Heart, Liver, Kidneys, Spleen, Testes and Adrenal glands were weighed prior to fixation.
HISTOPATHOLOGY: Yes. The following tissues were taken from each rat and preserved in 10% buffered formalin: Adrenal glands, Jejunum, Prostate, Aorta, Kidneys, Rectum, Bladder, Larynx, Sciatic nerve, Brain, Liver, Spinal cord, Caecum, Lungs, Spleen, Cervix, Lymph nodes (cervical and mesenteric), sternum, Colon, Mammary glands, Stomach, Duodenum, Muscle, Thymus, Femur and stifle joint, Oesophagus, Thyroid and parathyroids, Head, Ovaries and fallopian tubes, Tongue, Heart, Pancreas, Trachea, Ileum, Pituitary, Uterus.
The following tissues were taken from each rat and preserved in Bouin's fixative: Epididymides, Salivary glands, Seminal vesicles, Skin, Testes, Vagina.
The eyes/harderian glands were taken from each rat and fixed in Davidson's fluid.
Bone marrow smears were taken and stained with May-Grunwald Giemsa.
Microscopic examination was carried out on all of the tissues from all animals in the control and hig-dose groups, and on all tissues showing macroscopic abnormalities which were designated as lesions at necropsy. In addition, livers, adrenals, mesenteric lymph nodes, spleens and thyroids (females only) from all animals fed 1.0 and 0.1% test substance were examined microscopically following identification of treatment-related lesions in the high-dose group.
During the examination of tissues, histopathological changes were recorded as present or absent, or graded according to their morphology, using a numerical scale of 0.0 – 5.0 in order to assess degrees of severity or activity. This procedure provided a means of ranking degrees of change which can assist in the interpretation of biological differences.
Statistics:
Statistical analysis was conducted for data on body weight, food and water intake, food conversion efficiency, clinical pathology and organ weights. Initially the data were examined to see if parametric or non-parametric analysis was appropriate.
For parametric data, one way analysis of variance was used to assess differences. A t-test was used to show any significant differences between control and test groups at the 5, 1 and 0.1% probability levels. A multiple T test was used for pairwise comparisons between groups.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period.
Clinical signs generally involved scabs, alopecia, excoriation, nasal discharge and ocular discharge, and were not considered to be treatment-related as they were observed in all control and treatment groups.

BODY WEIGHT AND WEIGHT GAIN
No treatment-related changes in body weight (gain) were observed during the study period. Statistically significant changes in body weight were few, minor, randomly distributed and of no biological significance. Thus, the mean body weight of females fed 1% test material was slightly (3.7%) increased in Week 0. In the same group, body weight gain was slightly (7.6%) increased in the period comprising Week 0-4.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption was significantly decreased during the first four weeks of the study in males (-5.4%) and females (-8.4%) fed 5.0% test substance in diet. This may reflect an initial reluctance of the animals to eat the diet.

FOOD EFFICIENCY
Food conversion efficiency was significantly increased (9.5%) in females of the 5.0% group during the first four weeks of the study.

WATER CONSUMPTION
Statistically significant changes (increases and decreases) in water consumption were observed but there was no clear treatment-related pattern. The accumulated water consumption was not significantly different between groups.

OPHTHALMOSCOPIC EXAMINATION
At the end of the study, a persistent pupillary membrane was recorded for one male in the 0.1% group and ocular opacity was recorded for one female in the 5.0% group. These findings were not considered to be treatment-related.

HAEMATOLOGY (Table 2)
Mean cell haemoglobin was slightly (2.3%) but statistically significantly increased in females of the 5.0% group. Prothrombin time was likewise slightly but significantly increased in males of the 5.0% group (3%) and in females of the 1.0 and 5.0% groups (2.6 and 4.3%, respectively). The observed changes were slight and unlikely to be toxicologically relevant.
A number of other changes (e.g. decreased neutrophil counts in females fed 1 .0 and 5.0% test substance) were statistically significant by Student's t-test but did not trigger the multiple t-test as significant.

CLINICAL CHEMISTRY (Table 3)
The following statistically significant changes were observed but not considered to be toxicologically relevant, since they were minor (< 10%) and/or no dose-relationship was noted: decrease in plasma calcium (males at 5.0% and all female groups), glucose (females at 1.0%), total protein (males and females at 5.0%) and increase in albumin/globulin ratio (males at 1.0 and 5.0%).
Aspartate aminotransferase was increased in females of the 0.1 and 5.0% groups and serum albumin was increased in males and females of the 5.0% group, but without dose-relationship.
Similarly, no clear evidence for a dose dependency was seen for the decrease in 5’-nucleotidase (males at 0.1 and 5.0% and females at 5.0%) and the increase in alanine aminotransferase (males and females at 5.0%).
Bilirubin was statistically significantly increased in males of the 1.0 and 5.0% groups. However, the levels measured were below the sensitivity of the method used and must therefore be viewed with caution.
Total cholesterol and triglycerides were significantly and dose-dependently decreased in males and females of the 1.0 and 5.0% groups. A decrease in beta-globulin was observed in males of the 5% group.
The statistically significant and dose-dependent increase in alkaline phosphatase observed in males and females of the 1.0 and 5.0% groups was marked, the level being more than double that of the control group in animals fed 5.0% test substance.

ORGAN WEIGHTS (Table 4)
Absolute and relative spleen weights were statistically significantly reduced in males of the 1.0 and 5.0% groups. Kidney weights were slightly but significantly reduced in females of the 1.0% (only relative weight) and 5.0% (absolute and relative weights) groups. Absolute liver weight was significantly reduced in females of the 0.1% group, while the relative weights were significantly reduced in all female groups, but without any dose-relationship. Absolute and relative liver weights were slightly but significantly reduced in males of the 1.0 and 5.0% groups.
The observed effects had no relation to any effect which might have been expected on the basis of the histopathology findings.

GROSS PATHOLOGY (Table 5)
Treatment with the test substance had no effects on bodily condition as assessed by estimation of abdominal fat reserves at necropsy.
Mesenteric lymph nodes were slightly enlarged in 1/20 male and 2/20 females of the 0.1% groups, in 13/20 males and 14/20 females of the 1.0% groups and in 13/20 males and 16/20 females of the 5.0% groups. Moderate enlargement was seen in 1/20 males at 1.0% and in 7/20 males and 2/20 females at 5.0%.
The colour of caecal contents was affected by feeding with the test substance. Caecal contents were predominantly yellow in rats fed 5.0% test substance and predominantly yellow-green in rats given 1.0% test substance, compared with green or gray-green in rats fed 0.1% test substance or the control animals. It should be note that the test substance was a yellow liquid.
The incidence of uterine fluid distension was slightly increased in rats fed 5.0% test substance (15/20 animals) compared with the control (8/20 animals).
All other macroscopic findings were considered to be incidental and within the range expected for this age and strain of rat.

HISTOPATHOLOGY: NON-NEOPLASTIC (Table 6)
Microscopic examination revealed treatment-related findings in mesenteric lymph nodes, spleen, liver, adrenal glands and thyroid glands (in females). Only those effects seen in the mesenteric lymph nodes and spleen extended down to the group fed 0.1% test substance.

-Mesenteric lymph nodes:
Macrophage aggregation(s) (some containing a golden brown pigment) were noted in the paracortex and in the medullary cords in all rats fed 5.0% test substance, in 11/19 male and 17/19 female rats fed 1.0% test substance and in 3/18 male and 8/19 female rats fed 0.1% test substance. Incidence and number of aggregations were dose-related, with only a few aggregations being present in rats given 0.1% test substance. There was a correlation between histological findings in the mesenteric lymph nodes and lymph node enlargement seen at necropsy.

-Spleen:
Golden/dark brown pigmented macrophages were observed in the red and white pulp of all animals fed 5.0% test substance, 16/20 males and 19/20 females fed 1.0% test substance and 5/20 females fed 0.1% test substance. Incidence and amount of pigmented macrophages was dose-related in male and female rats, the effect being more pronounced in females.

-Liver:
In male rats fed 5.0% test substance, the incidence of bile duct proliferation was increased (18/20 animals compared to 5/20 animals in the control group), as was incidence of sclerotic bile ducts (13/20 animals compared to 3/20 animals in the control group). Sclerosis was associated with minimal infiltration of mixed inflammatory cells. There was a very slight increase in the incidence of bile duct proliferation in female rats given 5.0% test substance (5/20 treated compared to 2/20 control animals). Periportal cytoplasmic vacuolation was decreased in males and females given 1.0% and 5.0% test substance.

-Adrenals:
In female rats fed 5.0% or 1.0% test substance, cortical vacuolation was noted in the adrenal glands (13/20 and 20/20 rats, respectively). Trace levels of vacuolation were seen in one female fed 0.1% test substance. This finding was not considered toxicologically important as vacuolation may occasionally be seen in control female animals. Cytoplasmic rarefaction was decreased in females given 5.0% test substance (0/20 treated rats compared to 19/20 control rats).
Cortical extramedullary haemopoiesis was not present in females fed 5.0% test substance. The incidence of extramedullary haemopoiesis was slightly reduced in female rats fed 1.0% or 0.1% test substance (6/20 and 5/20 animals respectively compared to 10/20 control animals). However, this was not considered of toxicological importance given that the incidence of this finding generally varies considerably among groups of untreated animals.

-Thyroids:
A slight increase in follicular epithelial hypertrophy was observed in female rats fed 5.0% test substance (15/20 animals) when compared with the control (5/20 animals).

-Spontaneous pathology:
Microscopic examination of the uteri from rats showing macroscopic fluid distension revealed that this was due to a variety of different reasons: luminal dilatation or dilated/cystic endometrial glands. This finding is not thought to be of any toxicological importance, in view of this, and in view of the variation in uterine size with different phases of the estrous cycle.
The incidence of retinal folding/atrophy was higher in animals of the test groups. However, since the overall incidence was very low and no dose relationship was observed, these lesions were not considered toxicologically relevant.
There was a slight increase in the incidence of lesions in the nasal passage in rats of the 5.0% group compared with the control groups. Lesions were minor in nature and included focal epithelial hypertrophy or hyperplasia associated with mucosal inflammatory cells or a luminal inflammatory exudate. Lesions of this nature are a common finding in control rats, and, while it is possible that they could be exacerbated by inhalation of diet containing irritant test material, the incidence in treated rats was still within the normal range.
A variety of spontaneous changes was observed in rats from all test groups without evidence of a treatment-related distribution. Findings were within the spectrum of spontaneous lesions commonly seen in laboratory rats of this age and strain and were thus considered no to be related to the test substance.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 other: % in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: adaptive effects on clinical chemistry (increase in alkaline phosphatase, slight decrease in total cholesterol and triglycerides) and histopathology (pigmented macrophage aggregation in lymph nodes and spleen)
Dose descriptor:
NOAEL
Effect level:
ca. 741 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
average dose calculated on the basis of the reported body weight and food intake data
Sex:
male
Basis for effect level:
other: corresponding to 1% in diet based on reported mean body weight and food intake data
Dose descriptor:
NOAEL
Effect level:
ca. 855 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
average dose calculated on the basis of the reported body weight and food intake data
Sex:
female
Basis for effect level:
other: corresponding to 1% in diet based on reported mean body weight and food intake data

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Achieved dose levels.

Treatment

Body weight (g)

Mean body weight (g)

Food intake (g)

Mean food intake (g/day)

Mean compound intake (g/day)

Mean dose level (mg/kg bw/day)

Week 0

Week 13

Week 0-13

Week 0-13

Week 0-13

Week 0-13

Week 0-13

Males

5.0%

133.7

541.5

337.6

2182.3

24.2

1.21

3591.2

1.0%

135.1

533.5

334.3

2229.1

24.8

0.25

740.9

0.1%

135.8

560.8

348.3

2321.6

25.8

0.03

74.1

Control

133.3

545.5

339.4

2215.3

24.6

0.00

0.0

Females

5.0%

120.2

317.5

218.85

1609.4

17.9

0.89

4085.5

1.0%

123.6

335.9

229.75

1767.7

19.6

0.20

854.9

0.1%

120.2

317.7

218.95

1782.7

19.8

0.02

90.5

Control

119.2

317.5

218.35

1693.8

18.8

0.00

0.0

 

Table 2. Haematology

Treatment

Mean cell haemoglobin (pg)

Prothrombin time (s)

Males

5.0%

18.16*

11.90**

1.0%

17.74

11.65

0.1%

17.58

11.54

Control

17.75

11.55

Females

5.0%

18.45

11.92***

1.0%

18.57

11.73*

0.1%

18.51

11.58

Control

18.41

11.43

 

* Statistically significantly different versus control (P = 0.5)

** Statistically significantly different versus control (P = 0.1)

*** Statistically significantly different versus control (P = 0.01)

 

Table 3. Clinical chemistry.

Treatment

Calcium

(mmol/L)

5’-Nucleotidase

(U/L)

Alkaline phosphatase

(U/L)

Alanine aminotransferase

(U/L)

Aspartate aminotransferase

(U/L)

Triglycerides

(mmol/L)

Total cholestertol

(mmol/L)

Males

5.0%

2.502***

28.4***

810.6***

65.1***

104.5

0.618***

1.901***

1.0%

2.559

32.7

606.3***

52.8

107.3

1.247***

2.380*

0.1%

2.598

32.2*

424.7

40.4

95.1

1.701

2.517

Control

2.604

36.3

389.5

41.0

93.8

1.820

2.803

Females

5.0%

2.567***

47.0**

512.6***

43.1***

93.1***

0.475***

1.766***

1.0%

2.626**

63.4

393.3***

30.5

75.9

0.876

2.181*

0.1%

2.612***

74.3

250.4

29.1

81.5**

0.965

2.362

Control

2.694

76.3

216.4

29.6

71.1

1.120

2.441

 

Treatment

Glucose

(mmol/L)

Bilirubin

(µmol/L)

Total protein

(g/L)

Albumin

(g/L)

Albumin/globulin ratio

beta-globulin

(g/L)

Males

5.0%

9.353

3.1***

62.07***

29.23*

0.905**

10.64***

1.0%

9.815

2.7*

64.09*

30.44

0.905**

11.89

0.1%

10.258

2.5

65.44

30.16

0.860

12.29

Control

10.053

2.3

66.58

30.52

0.845

12.36

Females

5.0%

9.420

2.7

67.29**

33.76***

1.015

10.65

1.0%

9.373*

2.8

70.59

36.24

1.055

10.70

0.1%

9.993

2.6

71.10

35.97

1.025

11.15

Control

9.852

2.6

72.35

37.12

1.050

11.43

 

* Statistically significantly different versus control (P = 0.5)

** Statistically significantly different versus control (P = 0.1)

*** Statistically significantly different versus control (P = 0.01)

 

Table 4. Organ weights.

Treatment

Spleen

(g)

Spleen

(g/100 g bw)

Kidney

(g)

Kidney

(g/100 g bw)

Liver

(g)

Liver

(g/100 bw)

Males

 

 

 

 

 

 

5.0%

0.7520***

0.1381***

3.429

0.631

18.169*

3.343***

1.0%

0.8436*

0.1566*

3.441

0.641

18.739*

3.472**

0.1%

0.9571

0.1691

3.565

0.633

20.756

3.660

Control

0.9454

0.1723

3.635

0.663

20.610

3.737

Females

 

 

 

 

 

 

5.0%

0.5573

0.1765

2.028*

0.640**

10.781

3.395**

1.0%

0.5904

0.1764

2.178

0.650*

10.804

3.210***

0.1%

0.5808

0.1851

2.064

0.657

9.862***

3.125***

Control

0.6079

0.1924

2.167

0.688

11.520

3.640

 

* Statistically significantly different versus control (P = 0.5)

** Statistically significantly different versus control (P = 0.1)

*** Statistically significantly different versus control (P = 0.01)

 

 

Table 5. Incidence of macroscopic findings.

Macroscopic observation

Treatment

Control

0.1%

1.0%

5.0%

Males

No. of animals/group

20

20

20

20

Caecum

Contents green

5

6

4

0

Contents gray-green

15

13

0

0

Contents yellow

0

0

0

19

Contents yellow-green

0

1

16

1

Mesenteric lymph node

Enlarged (moderate)

0

0

1

7

Enlarged (slight)

0

1

13

13

Females

No. of animals/group

20

20

20

20

Caecum

Contents green

7

8

3

0

Contents gray-green

13

12

1

0

Contents yellow

0

0

0

20

Contents yellow-green

0

0

16

0

Mesenteric lymph node

Enlarged (moderate)

0

0

0

2

Enlarged (slight)

0

2

14

16

Uterus

Moderate fluid distention

1

4

6

3

Severe fluid distention

1

0

0

0

Slight distention

0

0

1

0

Slight fluid distention

8

7

6

15

 

Table 6. Incidence of microscopic findings.

Microscopic observation

Treatment

Control

0.1%

1.0%

5.0%

Males

Adrenals

No. examined

20

20

20

20

 

Without abnormalities

3

0

0

1

Adrenal cortex

Vacuolation

17

20

20

19

 

Extramedullary haemopoiesis

0

2

0

0

Thyroids

No. examined

20

0

0

20

 

Without abnormalities

10

0

0

8

 

Follicular epithelium hypertrophy

10

0

0

12

Mesenteric lymph node

No tissue available

0

2

1

0

 

No. examined

20

18

19

20

 

Without abnormalities

12

12

1

0

 

Pigmented macrophage aggregation(s)

0

1

11

20

 

Macrophage aggregation(s)

1

3

10

20

Spleen

No. examined

20

20

20

20

 

Without abnormalities

20

19

4

0

 

Pigmented macrophages

0

0

16

20

 

Extramedullary haemopoiesis

0

1

2

0

Liver

No. examined

20

20

20

20

 

Without abnormalities

0

0

1

0

 

Cytoplasmic vacuolation (periportal)

7

6

1

0

 

Bile duct proliferation

5

4

6

18

 

Sclerotic bile duct(s)

3

4

7

12

 

Mixed inflammatory cell infiltration (periportal)

1

1

1

11

Females

Adrenal cortex

No. examined

20

20

20

20

 

Without abnormalities

0

0

1

0

 

Vacuolation

0

1

13

20

 

Extramedullary haemopoiesis

10

6

5

0

 

Cytoplasmic rarefaction

19

16

14

0

Thyroids

Follicular epithelium hypertrophy

5

5

6

15

Mesenteric lymph node

No tissue available

0

1

1

0

 

No. examined

20

19

19

20

 

Without abnormalities

17

5

1

0

 

Pigmented macrophage aggregation(s)

0

3

9

19

 

Macrophage aggregation(s)

0

8

17

20

 

Focus(i) foamy macrophages

0

1

0

0

Spleen

No. examined

20

20

20

20

 

Without abnormalities

20

15

1

0

 

Pigmented macrophages

0

5

19

20

Liver

No. examined

20

20

20

20

 

Without abnormalities

0

1

5

5

 

Cytoplasmic vacuolation (periportal)

10

13

6

2

 

Bile duct proliferation

2

0

0

5

 

Applicant's summary and conclusion

Conclusions:
Based on clinical chemistry parameters and histopathological findings, 1.0 % (w/w) test material in diet can be considered a no-observed-adverse-effect-level (NOAEL), corresponding to a dose of 741 and 855 mg/kg bw/day for males and females, respectively.