Fatty acids, C18-unsatd., dimers, di-Me esters, hydrogenated

Repeated dose toxicity: oral

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 were conducted; most but not all haematological/clinical chemistry, organ weight and histopathological examinations were conducted.

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

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

no functional observations were conducted; most but not all haematological/clinical chemistry, organ weight and histopathological examinations were conducted

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18-unsatd., dimers

EC Number:

500-148-0

EC Name:

Fatty acids, C18-unsatd., dimers

Cas Number:

61788-89-4

IUPAC Name:

61788-89-4

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: 73% dimer acid; 21% trimer acid
- Source: Unichema, Gouda; Holland

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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 (males); 104.6-142.0 g (females)
- Housing: groups of 5 animals per 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): the experimental diets were initially prepared on a weekly basis from 26 Nov - 10 Dec 1992. After data was obtained confirming stability of the test material in diet for 14 days, diets were prepared biweekly 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 room temperature before entry in the SPF unit, where they were stored at ca. 4 °C before use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

-Stability and homogeneity in diet
Stability of the test substance at 0.1%, 1% and 5% in diet was determined over periods of 7 and 14 days when stored at 4°C or in the animal room.
Diets were analysed in order to confirm homogeneous test substance distribution in the diet. After diet mixing, 5 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 / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.1, 1, 5% (w/w)
Basis:
nominal in diet

Doses / concentrations 2

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

Doses / concentrations 3

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 per day (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 once per week 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. Food intake values were determined two times per week and weekly values were calculated for each cage of 5 animals,
- 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 consumption values were determined two times per week and weekly values were calculated for each cage of 5 animals,

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment 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 treatment 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.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All surviving animals 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. The following organs were weighed prior to fixation: brain, heart, liver, kidneys, spleen, testes and adrenal glands.
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):

refer to Details on results

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

refer to 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):

refer to Details on results

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

refer to 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):

refer to Details on results

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

refer to Details on results

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

refer to Details on results

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the study period.
Clinical signs generally involved scabs, alopecia, excoriation, nasal discharge and ocular discharge, and were considered to be not treatment-related, since they were observed in all groups (control and treatment).

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

FOOD CONSUMPTION AND COMPOUND INTAKE
There was a significant decrease in food consumption during the first 4 weeks of the study in males (-5.4%) and females (-8.4%) in the 5.0% group, which may reflect an initial reluctance of the animals to eat the diet.

FOOD EFFICIENCY
There was a significant increase in food conversion efficiency (9.5%) during the first four weeks of the study in females of the 5.0% group.

WATER CONSUMPTION
There were statistically significant changes (increases and decreases) in water consumption but without a clear treatment-related pattern. Accumulated water consumption was not significantly different between groups.

OPHTHALMOSCOPIC EXAMINATION
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 at the end of the study. These findings were considered to be not treatment-related.

HAEMATOLOGY (Table 2)
In the 5.0% group, mean cell haemoglobin was slightly (2.3%) but statistically significantly increased in females; prothrombin time was likewise slightly but significantly increased in males (3%) and females (4.3%).
In the 1.0% group, prothrombin time was slightly but significantly increased in females (2.6%).
The observed changes were slight and unlikely to be toxicologically relevant.
A number of other changes (e.g. decreased neutrophil counts in females of the 1 .0 and 5.0% groups) 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 (5.0% male group and all female groups), glucose (1.0% female group), total protein (5.0% male and female groups) and increase in albumin/globulin ratio (1.0 and 5.0% male groups).
Aspartate aminotransferase was increased in females fed 0.1 and 5.0% test item and serum albumin was increased in males and females given 5.0% test item, but there was no dose-relationship.
Likewise, there was no clear evidence for a dose dependency for the decrease in 5’-nucleotidase (0.1% male groups and 5.0% male and female groups) and the increase in alanine aminotransferase (males and females of the 5.0% group).
Bilirubin was statistically significantly increased in males fed 1.0 and 5.0% test item in diet. 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 at 1.0 and 5.0% in males and females. There was a decrease in beta-globulin in males of the 5.0% 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 determined level was more than double that of the control group in animals of the 5.0% group.

ORGAN WEIGHTS (Table 4)
There was a statistically significant reduction in absolute and relative spleen weights in males of the 1.0 and 5.0% groups. There was a slight but statistically significant reduction in kidney weights in females fed 1.0% (only relative weight) and 5.0% (absolute and relative weights) test item in diet. In females of the 0.1% group, absolute liver weight was significantly reduced, while relative weights were significantly reduced in all female groups, but without showing any dose-relationship. Absolute and relative liver weights were slightly but significantly reduced in the 1.0 and 5.0% male 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)
There were no treatment-related effects on bodily condition as assessed by estimation of abdominal fat reserves at necropsy.
Slightly enlarged mesenteric lymph nodes were noted in 1/20 males 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 observed 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 animals of the 5.0% group, predominantly yellow-green in animals of the 1.0% group, and green or gray-green in animals of the 0.1% or the control group, respectively. It should be note that the test substance was a yellow liquid.
There was a slightly increased incidence of uterine fluid distension in animals fed 5.0% test item (15/20 animals) compared with the control group (8/20 animals).
All other macroscopic findings were considered to be incidental and within the range expected for this age and strain of rats.

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 animals of the 5.0% group, in 11/19 male and 17/19 females of the 1.0% group and in 3/18 male and 8/19 female animals of the 0.1% group. The incidence and number of aggregations were dose-related, and there were only a few aggregations present in animals of the 0.1% group. There was a correlation between histological findings in the mesenteric lymph nodes and lymph node enlargement noted at necropsy.

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

-Liver:
In male animals of the 5.0% group, there was an increased incidence of bile duct proliferation (18/20 animals vs. 5/20 animals in the control group) and sclerotic bile ducts (13/20 animals vs. 3/20 animals in the control group). Sclerosis was associated with minimal infiltration of mixed inflammatory cells. A very slight increase in the incidence of bile duct proliferation was seen in females of the 5.0% group (5/20 treated animals vs. 2/20 control animals). Periportal cytoplasmic vacuolation was decreased in males and females fed 1.0% and 5.0% test substance in diet.

-Adrenals:
In female animals of the 5.0% or 1.0% groups, cortical vacuolation was seen in the adrenal glands (13/20 and 20/20 rats, respectively). Trace levels of vacuolation were noted in one female of the 0.1% group. This finding was considered to be not toxicologically important, since vacuolation may occasionally be seen in control female animals. Cytoplasmic rarefaction was decreased in females fed 5.0% test item in diet (0/20 treated animals vs. 19/20 control rats).
Cortical extramedullary haemopoiesis was not present in females of the 5.0% group. A slightly reduced incidence of extramedullary haemopoiesis was noted in females given 1.0% or 0.1% test item in diet (6/20 and 5/20 animals, respectively, vs. 10/20 control animals). However, this was considered to be of no toxicological importance since the incidence of this finding generally varies considerably among groups of untreated animals.

-Thyroids:
A slight increase in follicular epithelial hypertrophy was noted in females of the 5.0% group (15/20 animals vs. 5/20 control animals).

-Spontaneous pathology:
Microscopic examination of the uteri from rats showing macroscopic fluid distension showed that this finding was due to a variety of different reasons: luminal dilatation or dilated/cystic endometrial glands. This finding was 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 treated rats. However, given that the overall incidence was very low and there was no dose relationship, these lesions were not considered to be toxicologically relevant.
A slight increase in the incidence of lesions in the nasal passage was observed in animals of the 5.0% group when 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 animals was still within the normal range.
A variety of spontaneous changes was noted in animals of all treatment 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 therefore considered to be not substance-related.

Effect levels

1

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)

2

Dose descriptor:

NOAEL

Effect level:

741 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: corresponding to 1% in diet based on reported mean body weight and food intake data

3

Dose descriptor:

NOAEL

Effect level:

855 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: corresponding to 1% in diet based on reported 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:

1.0 % (w/w) test material in diet can be considered a NOAEL based on clinical chemistry parameters and histopathological findings, this corresponds to a dose level of approximately 741 and 855 mg/kg bw/day for male and female rats, respectively.