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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
Study period:
From 4 August 1999 to 3 September 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study with acceptable restrictions. The analytical purity of the test substance was not specified. The test compound tested is the 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate - EC number: 261-665-2 | CAS number: 59219-71-5

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001
Reference Type:
publication
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
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): 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate
- Analytical purity: no data
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 98J459
- Expiration date of the lot/batch: not specified
- Purity test date: At finalization of the study report, no analytical certificate was available. Characterisation of the test substance, which appropriately defines the tested batch is under the responsability of the Sponsor.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle:At finalization of the study report, no analytical certificate was available. Characterisation of the test substance, which appropriately defines the tested batch is under the responsability of the Sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance was administered as a solution in the vehicle. The test substance was diluted with the required quantity of vehicle in order to achieve the concentration of 20, 60 and 200 mg/mL and then homogenized using a magnetic stirrer. The test substance dosage forms were made daily.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen because it is a rodent species commonly requested by the international regulations for this type of study. The Sprague Dawley strain was selected because background data from previous studies was available at the laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: six weeks old
- Weight at study initiation: male : 204g (187 to 226g) / female : 169g (154 to 186g)
- Fasting period before study: not specified
- Housing: housed in suspended wire-mesh cages (43x21.5x18 cm)
- Diet (e.g. ad libitum): A04C pelleted maintenance diet (UAR France) ad libitum
- Water (e.g. ad libitum): tap water filtered using a 0.22 micron filter ad libitum
- Acclimation period: at least seven days

DETAILS OF FOOD AND WATER QUALITY: Bacterial and chemical analysis of diet and water were performed, including pesticides, heavy metals and nitrosamines. No contaminants were known to be present in the diet and water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2 deg Celsius
- Humidity (%): 50±20% relative humidity
- Air changes (per hr): approximately 12 cycles/hour of filtered, non recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h (07:00-19:00)

IN-LIFE DATES: From: 4 August 1999 To: 3 September 1999

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since it is expected to ensure an absorption of the test substance at least equal to the cutaneous route which is the route of exposure in human.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was administered as a solution in the vehicle. The test substance was diluted with the required quantity of vehicle in order to achieve the concentration of 20, 60 and 200 mg/mL and then homogenized using a magnetic stirrer. The test substance dosage forms were made daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification
- Concentration in vehicle: 20, 60, 200 mg/mL of test item in vehicle
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): No. 107H1649 (Sigma, France)
- Purity: Not specified
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 Days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Only Vehicle
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals were used per sex per dose.
Control animals:
yes
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): performed according to a computerized stratification procedure in order to have a similar average body weight in each group
- Rationale for selecting satellite groups: not satellite group
- Post-exposure recovery period in satellite groups: no satellite group
- Section schedule rationale (if not random): not specified
Positive control:
No positive control was used

Examinations

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

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded once before the allocation of the animals to the groups, on the first day of treatment and then once a week until the end of the study.

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: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
Calculated as mg food/animal/day

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:Blood samples were taken before the daily treatment from the orbital sinus if the animals under light isofluorane anesthesia and collected into tubes containing the appropriate anticoagulant.
- Anaesthetic used for blood collection: Yes (light isofluorane)
- Animals fasted: Yes, overnight of at least 14 hours
- How many animals: 80
- Parameters checked - Parameters checked : Erythrocytes (RBC), Haemoglobin (HB), mean cell volume (MCV), Packed cell volume (PCV), mean cell haemoglobin concentration (MCHC), mean cell haemoglobin (MHC), thromocytes (PLAT), leucocytes (WBC), differential white cell count with cell morphology (neutrophils, eosinophils, basophils, lymphocytes and monocytes) and reticulocytes (RETIC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Bloods were taken (before treatment) from the orbital sinus of the animal under slight isoflurane anesthesia and collected into tubes containing the appropriate anticoagulant. Haematologic parameters were determined day 9 for high dose group and at the end of the week 2 for the other groups
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes , overnight
- How many animals: 80 animals
- Parameters checked : Sodium, Potassium, Chloride, Calcium, Inorganic Phosphorus, Glucose, Urea, Creatinine, Total Bilirubin, Total Proteins, Albumin, Albumin/globulin ratio, Cholesterol, Triglycerides, Alkaline Phosphatase, Aspartate aminotransferase, Alanine aminotransferase

URINALYSIS: Yes
- Time schedule for collection of urine: The following parameters were determned for all animals at the end of the treatment period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Urine Volume, pH, specific gravity , proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, cytology of sediment, leucocytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals, cells., appearance and color of urine.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A complete macroscopic examination post mortem was performed on all animals of the study. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

Weighed Organs : Adrenals, Heart, Kidney, Liver, Ovaries, Spleen, Testes, Thymus, Thyroid

Preservation of tissue : Adrenals, Aorta, Brain, Caecum, Colon, Duodenum, Epididymes, Esophagus, Eyes with Harderian glands, Femoral bone with articulation, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs with bronchi, Lymph nodes, Mammary gland, Prostate, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord, Spleen, Sternum with Bone Marrow, Stomach with Forestomach, Testes, Thymus, Thyroid and Parathyroids, Tongue, Trachea, Urinary bladder, Uterus, Vagina

HISTOPATHOLOGY: Yes
All tissues required for microscopic examination were embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin.
Organs : Adrenals, Brain, Caecum, Colon, Duodenum, Epididymes, Esophagus, Eyes with Harderian glands, Femoral bone with articulation, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs with bronchi, Lymph nodes, Mammary gland, Prostate, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles, Skin, Spinal cord, Spleen, Sternum with Bone Marrow, Stomach with Forestomach, Testes, Thymus, Thyroid and Parathyroids, Trachea, Urinary bladder, Uterus, Vagina
Statistics:
Statistical method was details on the attached figure below.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was note in 5 males and all females given 1000 mg/kg/day, occasionally or up to the end of the study, after at least 6 days of treatment. Areas of hair loss, scattered hair and/or diffuse alopecia, were noted in one female given 100 mg/kg/day, in 3 females of the 300 mg/kg/day group, in 3 males and 7 females of the high dose group.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female died in the group given 300 mg/kg/day. Four females of the high dose level group was found dead prematurely. No mortality occurred at 100 mg/kg/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No marked differences were noted in bodyweight gain between control animals and animals given 100 or 300 mg/kg/day. When compared to the mean control value, a lower mean body weight gain was noted in males and females given 1000 mg/kg/day, during the whole treatment period. This difference was especially marked in week 1 and correlated with lower food consumption. A body weight loss was noted in week 4 in both males and females. This effect, correlated with lower food consumption, was attributed to treatment with the test substance.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
When compared to the mean control values, a higher mean monocyte level was noted with dose-relationship in all treated males and females. however, almost of all individual values remained within the range of historical data.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
When compared to mean control values, the following differences were noted at the end of the treatment period:
-higher Aspartate aminotransferase and Alanine aminotransferase activities (associated in females with higher Alkaline Phosphatase activity) in females given 300 mg/kg/day and all animals given 1000 mg/kg/day was measured.
-a lower mean glucose in all treated animals, almost all the individual values remained within the range of historical data values. This effect could be related to describe below changes observed in liver.
-a higher urea level in all treated males and females was observed which was in the range of historical data.
-a higher mean inorganic phosphorus level in females given 100 or 300 mg/kg /day and in males and females given 1000 mg/kg /day were noted. Due to low control values, the higher mean inorganic phosphorus values was not considered as toxicological relevant, there was not correlated with ionic changes and all individual datas were within the historical data.

Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The only difference noted was a statistically higher volume of urine in females given 100 mg/kg/day, and in males and females of the high dose group. This was correlated with a lower specific gravity. The higher urine volume for the high dose level group was considered to be related to the treatement and could be related to the renal changes observed among these animal (described below).
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The higher kidneys weights observed in the males were considered treatment related, could be correlated to the acidophilic globules in the cortical tubular epithelium of the kidneys noted at microscopic examination (section histopathological findings : non-neoplastic section). In females of the 300 and 1000 mg/kg/day dose groups, a higher kidney weights was observed.
Higher dose related liver weights were observed in two sexes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Grey/green colouration of the kidneys were noted in one male and one female of the group given 100 mg/kg/day, in two females of the intermediate dose group, in five male and one female of the 1000 mg/kg/day group. This effect was related to males only, with the acidophilic globules in the cortical tubular epithelium.
Liver enlargement was noted in one male given 100 mg/kg/day, in one male and female given 300 mg/kg/day, in 6 males and 5 females of the 1000 mg/kg/day dose group. Accentuated lobular pattern was noted in 4 males and 7 females given 100 mg/kg/day, in 4 males and 9 females given 300 mg/kg/day, in 8 males and 3 females of the high dose group. Liver paleness was noted in 4 females given 100 mg/kg/day, in 3 females given 300 mg/kg/day, in 2 males and 7 females of the high dose group. All this effects on the liver were correlated with minimal to severe steatosis observed and/or slight to severe hepatocellular hypertrophy observed in microscopic examination.
Reduced spleen (1 female of the 300 mg/kg/day group, in 1 female and 4 males of the high dose group) ,reduced thymus (2 females given 1000 mg/kg/day) observed were considered to be secondary to the changes which occured on the liver and kidneys.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal to severe steatosis (perilobular, perilobular and mediolobular, or diffuse)was seen in 5 control females, in 5 males of the 100 mg/kg/day group, 7 males of the intermediate dose group, 9 males given 1000 mg/kg/day and all the treated females. Slight to severe hepatocellular hypertrophy was seen in 9 males of 300 and 1000 mg/kg/day groups and in all females treated with this doses.
Minimal to slight acidophilic globules in cortical tubular epithelium were noted in 3 control males. Slight to severe acidophilic globules in the cortical tubular epithelium were noted in all males of all the test substance treated groups. The higher incidence and severity of the acidophilic globule was considered to be caused by the increased production of alpha-2-microglobulin. This effect is only observed in male rodents.
Reduced spleen (1 female of the 300 mg/kg/day group, in 1 female and 4 males of the high dose group) ,reduced thymus (2 females given 1000 mg/kg/day) observed were considered to be secondary to the changes which occured on the liver and kidneys.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
clinical biochemistry
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
clinical biochemistry
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Table 1 : Summary of the results

Male

Female

 

 

Male

 

 

 

Female

 

 

 

 

Dose-Level (mg/kg/day)

0

100

300

860

0

100

300

860

Bodyweight

BodyweightGain (g)

151

143

131

106

72

81

68

43

 

Body weight change from day 1

1,7

1,7

1,6

1,5

1,4

1,5

1,4

1,3

 

Bodyweight at the end of the study

355

346

337

310**

240

250

235

214

 

Differencefromcontrol (%)

 

-3

-5

-13

 

-4

-2

-11

Foodconsumption

Food consumption (g/day/animal)

23,2

22,5

22,9

21,9

17,3

17,8

17,8

16,2

 

Differencefromcontrol (%)

 

-3

-5

-13

 

-4

-2

-11

Blooodbiochemistry

Urea

M

3,5

4,1

4,5**

4,3*

5,1

6

6,5*

6,6*

 

SD

0,59

0,59

0,63

0,48

0,48

1,1

1,6

1,02

 

Cholesterol

M

1,4

1,4

1,3

1,7

1,9

1,6

1,5

1,4*

 

SD

0,24

0,36

0,24

0,42

0,19

0,28

0,44

0,34

 

Triglycerides

M

0,63

0,53

0,55

0,75

0,44

0,67

0,51

0,67

 

SD

0,273

0,24

0,339

0,539

0,17

0,415

0,267

0,483

 

Alkalinephosphataseactivity

M

313

312

279

338

190

244

316**

364**

 

SD

35,6

62,6

51,4

111,8

40,4

73,5

118,7

158,9

 

AspartateAminotransferase

M

57

56

69

86**

59

50

75

120**

 

SD

6,3

7,2

13,9

29,1

15,2

18,9

17,9

41,1

 

AlanineAminotransferaseactivity

M

14

18

21

42**

14

24

35**

48**

 

SD

2,2

4,5

8

25,9

5,3

10,1

12,3

15,8

 

Glucose

M

7,17

5,85**

5,50**

5,50**

7,08

6,13*

5,70**

5,81*

 

 

SD

0,592

0,436

0,19

0,684

0,806

0,489

0,885

1,193

Organweight(g)

Liver

Absolute

 

2,7

18,8

47,8

 

32,4

55,1

85,8

 

Relative

 

6,7

28,1

71,9

 

29,4

64,5

114,7

 

Kidney

Absolute

 

17,9

14,5

13,7

 

12,3

3,4

6,7

 

Relative

 

22,8

22,7

31,1

 

10,1

9,4

23,5

 

Spleen

Absolute

 

-3,8

-10,9

-23

 

0,7

-20,1

-37,6

 

Relative

 

-0,4

-4,7

-10,7

 

-1,6

-15,1

-28,8

 

Thymus

Absolute

 

-3,6

-9,2

-23,1

 

-0,3

-20,6

-37

 

 

Relative

 

-0,4

-2,6

-9,2

 

-2,5

-16

-29,7

Severity of steatosis (number of animals)

Grade 1

0

0

0

0

4

0

0

1

 

Grade 2

0

4

2

3

1

4

1

1

 

Grade 3

0

1

2

1

0

1

1

1

 

Grade 4

0

0

3

4

0

0

8

3

 

Grade 5

0

0

0

0

0

0

0

4

 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the study, the test substance induced mortality at 300 and 1000 mg/kg/day, signs of kidney and liver steatosis all dose levels (grading slight to severe steatosis) with hepatocellular hypertrophy (grading slight to severe). This hepatic steatosis observed was present in all groups including control group (5 females) due to high fat load in liver. Indeed the vehicle, corn oil, was used for steatosis model in rats (CIR comments) and the effect noted was related to physiological metabolism of fatty acid in liver. This changes in liver became pathological in case of irreversible dysregulation of metabolism seen in higher activity of Alkaline phosphatase, Alanine transferase activities and lower mean value of plasmatic cholesterol (1000 mg/kg/day dose group). This effects was correlated to higher severity grading of steatosis at this two dose levels. In regard with the steatosis induced by control vehicle, the test item exacerbated the changes induced by corn oil. However, this changes was not adverse at control condition and at 100 mg/kg/day, there was no dysregulation of enzymatic activities and no variation of lipidemy in plasma (seen in plasmatic cholosterol and triglyceride concentration) and did not led to mortality (observed at 300 and 1000 mg/day/kg groups). Lower glycemia was observed in all tested animal and was considered as non adverse because values were within the range of historical data of the laboratory.
The No Observed Adverse Effect NOAEL in rats for the test substance in rat treated by oral route was defined at 100 mg/kg/day, the steatosis at this dose was not considered as pathological and adverse, no enzymatic dysregulation was observed, no higher fatty acid in blood was noted, the changes of liver was not considered as irreversible.
Executive summary:

The purpose of this GLP-compliant study was to evaluate the potential toxicity of the test substance Isononyl Isononanoate when daily administered orally in rats during 28 days according to the OECD Guideline 407 method.

Four groups of 20 Sprague Dawley rats (composed with 10 males and 10 females) were used in this repeated dose toxicity test by gavage route. They were exposed to different doses as 0, 100, 300 and 1000 mg/kg/day. The test substance was diluted in corn oil. The control group only received vehicle. During treatment period, different parameters were measured. The mortaility, clinical signs were checked daily. Body weight and food consumption were recorded once a week.Haematological, blood biochemical and urinalysis were performed the last day of the treatment period.On completion of the treatment period, animal were sacrified by CO2 asphyxiation and blood exsanguination. Macroscopic examination of tissues, with organ weighing and microscopic examination were performed on each animal (on survived animals and animals which died prematurely).

No marked differences were noted in bodyweight gain between control animals and animals given 100 or 300 mg/kg/day. When compared to the mean control value, a lower mean body weight gain was noted in males and females given 1000 mg/kg/day, during the whole treatment period. This difference was especially marked in week 1 and correlated with lower food consumption. A body weight loss was noted in week 4 in both males and females. This effect, correlated with lower food consumption, was attributed to treatment with the test substance.

Ptyalism was note in 5 males and all females given 1000 mg/kg/day, occasionally or up to the end of the study, after at least 6 days of treatment. Areas of hair loss, scattered hair and/or diffuse alopecia, were noted in one female given 100 mg/kg/day, in 3 females of the 300 mg/kg/day group, in 3 males and 7 females of the high dose group.

One female died in the group given 300 mg/kg/day. Four females of the high dose level group was found dead prematurely. No mortality occurred at 100 mg/kg/day.

When compared to mean control values, the following differences were noted at the end of the treatment period:

-higher Aspartate aminotransferase and Alanine aminotransferase activities (associated in females with higher Alkaline Phosphatase activity) in females given 300 mg/kg/day and all animals given 1000 mg/kg/day was measured.

-a lower mean glucose in all treated animals, almost all the individual values remained within the range of historical data values. This effect could be related to describe below changes observed in liver.

-a higher urea level in all treated males and females was observed which was in the range of historical data.

-a higher mean inorganic phosphorus level in females given 100 or 300 mg/kg /day and in males and females given 1000 mg/kg /day were noted. Due to low control values, the higher mean inorganic phosphorus values was not considered as toxicological relevant, there was not correlated with ionic changes and all individual datas were within the historical data.

Grey/green colouration of the kidneys were noted in one male and one female of the group given 100 mg/kg/day, in two females of the intermediate dose group, in five male and one female of the 1000 mg/kg/day group. This effect was significant for males only, with the acidophilic globules in the cortical tubular epithelium.

Liver enlargement was noted in one male given 100 mg/kg/day, in one male and female given 300 mg/kg/day, in 6 males and 5 females of the 1000 mg/kg/day dose group. Accentuated lobular pattern was noted in 4 males and 7 females given 100 mg/kg/day, in 4 males and 9 females given 300 mg/kg/day, in 8 males and 3 females of the high dose group. Liver paleness was noted in 4 females given 100 mg/kg/day, in 3 females given 300 mg/kg/day, in 2 males and 7 females of the high dose group. All this effects on the liver were correlated with minimal to severe steatosis observed and/or slight to severe hepatocellular hypertrophy observed in microscopic examination.

Reduced spleen (1 female of the 300 mg/kg/day group, in 1 female and 4 males of the high dose group) ,reduced thymus (2 females given 1000 mg/kg/day) observed were considered to be secondary to the changes which occurred on the liver and kidneys.

Under the experimental conditions of the study, the test substance induced mortality at 300 and 1000 mg/kg/day, signs of kidney and liver steatosis all dose levels (grading slight to severe  steatosis) with hepatocellular hypertrophy (grading slight to severe). This hepatic steatosis observed was present in all groups including control group (5 females) due to high fat load in liver. Indeed the vehicle, corn oil, was used for steatosis model in rats (CIR comments) and the effect noted was related to physiological metabolism of fatty acid in liver. This changes in liver became pathological in case of irreversible dysregulation of metabolism seen in higher activity of Alkaline phosphatase, Alanine transferase activities and lower mean value of plasmatic cholesterol (1000 mg/kg/day dose group). This effects was correlated to higher severity grading of steatosis at this two dose levels. In regard with the steatosis induced by control vehicle, the test intem exacerbated the changes induced by corn oil. However, this changes was not adverse at  control condition and at 100 mg/kg/day, there was no dysregulation of enzymatic activities and no variation lipidemy in plasma (seen in plasmatic cholosterol and triglyceride concentration) (observed at 300 and 1000 mg/day/kg groups). Lower glycemia was observed in all tested animal and was considered as non adverse because values were within the range of historical data of the laboratory.

The No Observed Adverse Effect NOAEL in rats for the test substance in rat treated by oral route was defined at 100 mg/kg/day, the steatosis at this dose was not considered as pathological and adverse, no enzymatic dysregulation was observed, no  higher fatty acid in blood was noted, the changes of liver was not considered as irreversible.