Registration Dossier

Administrative data

Description of key information

According to the available key study (Manciaux, 2001, GLP, OECD Guideline 407 Method, Klimisch2), the test substance monoconstituent 3,5,5 trimethylhexyl 3,5,5-trimethylhexanoate (RN CAS: 59219-71-5) when administered on rats daily during 28 days by gavage at 0, 100, 300 and 1000 mg/kg/day, induced exacerbation of effects induced by vehicle corn oil on liver (effect induced by the high fat load in liver by administration of the vehicle and the test item). Indeed, some animals of each group (including control) showed hepatic steatosis. This hepatic effect was considered to be pathological and irreversible at the dose of 300 mg/kg/day (due to dysregulation of enzymatic activities and aggravated hepatic steatosis). Hence the No Observed Effect Level was defined at 100 mg/kg/day and the Low Observed Adverse Effect Level at 300 mg/kg/day based on hepatic toxic effect. Considering the dose effect, the severity of the effects which refers to known mechanism of fatty acid. According to the CLP criteria, the substance should not be classified with a STOT.

3 studies were available to estimate the repeated dose toxicity by dermal route. No relevant NOAEL or LOAEL could be defined but similar systemic toxic effects than by oral route were reported plus skin irritation effects that were observed after repeated dermal exposure. The results indicated a probable hydrolysis of the ester in the skin and after oral route in systemic compartment. The toxic effects observed seems due to the isononanoic acid formed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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.
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
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
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
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.
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
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
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

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

 

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
hepatobiliary
Organ:
liver

Additional information

One key study was available to assess the potential toxicity of the test item : Manciaux, 2001, GLP, OECD Guideline 407 Method, Klimisch 2.

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 monoconstituent 3,5,5 trimethylhexyl 3,5,5-trimethylhexanoate (RN CAS: 59219-71 -5) 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 analysis 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).

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.

Macroscopic and microscopic examinations mainly showed hepatic effect by liver enlargement and hepatocellular hypertrophy correlated with hepatic steatosis, in animals of each group including control. (25% of rats in control group, 75% at 100mg/kg/day, 85% at 300 mg/kg/day, 90% of the high dose level group). From the dose of 300 mg/kg/day, higher activites of enzymatic activity was observed as Aspartate Aminotransferase, Alanine Aminotransferase and Alkaline Phosphatase activity and lower plasmatic cholesterol rate (only in the high dose group). At his doses, observation of changes in thymus and spleen was correlated with previous described changes in liver.

Three studies by dermal route had been performed on rats.

1) In a repeated dose toxicity by dermal routes on sprague dawley rats according to OECD Guideline 410 method, test item monoconstituent 3,5,5 trimethylhexyl 3,5,5-trimethylhexanoate (RN CAS: 59219-71-5) was administered to 40 Sprague Dawley rats.

5 males and 5 females by groups were exposed dermally to the test item (diluted in corn oil) at 100, 300 and 800 mg/kg/bw, during 8 days for the high dose groups and 15 days for the other groups. The animals were checked daily for mortality, morbidity and clinical signs. Body weight and food consumption were recorded twice a week. Hematological and blood chemical investigations were performed on all animals at the end of the treatment period (8 days or two weeks). On the completion of the treatment period, animals were submitted to a complete macroscopic post mortem examination and selected organs were weighed. Macroscopic lesions and specified tissues were preserved. Microscopic examination was performed for specified groups on selected tissues or lesions.

No mortality occured during the study. However, due to ethical grounds, the high dose group was sacrified the day 8 (severe cutaneous reactions). Sligh cutaneous reactions were noted at 100 and 300 mg/kg/day (one animal in each group). Severe irritation and necrosis (severe erythema, cutaneous lesions and desquamation) were observed for animals of the high dose level group.

A lower body weight gain or a body weight loss, associated with a lower food consumption was noted for this group.

Low blood cell, eosinophils and lymphocytes count, and neutrophil leucocytosis were noted for the group given 860 mg/kg/day, considered to be the consequence of the inflammatory reaction at the application site.

For blood biochemistry; at 860 mg/kg bw/day, when compared to the range of historical data, the following changes were noted :

-a high mean urea level

-a low mean total protein level

-a low mean total cholesterol

-a high mean ALP (alkaline phosphatase) activity

- a high mean aspartate aminotransferase activity

At 100 and 300 mg/kg/day, when compared to the mean control values, the following changes were

noted :

-a higher mean urea level in males and females given 300 mg/kg/day

-a lower mean cholesterol level in female given 100 mg/kg/day and in males and females given 300 mg/

kg/day, almost all the individual values were outside the range of historical data

-a higher mean triglycerides level in males and females given 100 mg/kg/day

-a higher mean Alkaline phosphatase activity in females given 300 mg/kg/day

-a lower mean protein level in females given 300 mg/kg/day

Scrabs on the treated skin, associated with ulceration, degeneration or necrosis of the epidermis, inflammation of the dermis and acanthosis, were noted in all animal of the high dose group. Accentuated lobular pattern associated at microscopic examination with hepatocellular hypertrophy and steatosis was noted in some animals given 300 or 860 mg/kg/day. Cortical cell hypertrophy of the adrenal glands was noted in the animals given 860 mg/kg/day.

Under the experimental conditions of the study, the test item 3,5,5 trimethylhexyl 3,5,5 -trimethylhexanoate induced toxicity at all dose level used in this study, as hepatic steatosis, haematological changes in inflammatory reaction and cutaneous irritation. The No Observed Adverse Effect NOAEL cannot be defined. Given the highly lipophilic property of the substance (logP=7.37), the dermal penetration should be very low (overestimation of 10% according to CIR opinion). It was possible that the water used for washing did not removed completely the test substance and the corn oil used. The toxic effects observed on the animals as (hepatic steatosis, hepatocellular hypertrophy, and cholesterol and triglycerides dyregulation were seen in an other repeated dose toxicity study (Manciaux, 2001)). It was possible that a secondary exposure route occurred in this study by grooming and licking the application site, the rats did not have a restrainer to prevent the ingestion during the treatment period.

This study cannot be used to assess the only dermal toxicity of the test substance.

2) A dose range finding and an OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test were performed on rats. However, due to the clinical condition of the animals, including moribundity and adverse clinical findings, dosing was discontinued following 8 days of exposure, and the entire study was terminated after up to an 11-day non-dosing period.

Dermal application of the pure test item (applied neat) at dose of 400 or 800 mg/kg bw/d resulted in the moribundity and subsequent euthanasia in extremis of 5 males and 1 female in the 800 mg/kg bw/day group. In this study, dermal application of the neat test item caused marked body weight losses and damage to the skin resulting in very slight to moderate erythema, very slight to slight edema, fissuring, desquamation, and exfoliation resulting in the early termination of this study. At the scheduled necropsies (study days 10-11 and study day 17), alterations in hematological and clinical chemistry panels were noted in combination with gross macroscopic and microscopic findings in a variety of organs and tissues. These effects indicated injury at the dermal application site and inflammatory effects due to exposure to the test item, but were partially reversible following a 10- or 11-day non-dosing period.

Based on these results, dermal application of the neat test item at 400 and 800 mg/kg bw/day was not considered appropriate for daily exposure to rats. A NOAEL or LOAEL could not be derived.

Therefore, to meet the standard information requirements of Regulation (EC) 1907/2006, Annex IX, Column 1, 8.6.2, a GLP-compliant subchronic (90-day) toxicity study in the rat via the oral route following OECD 408 with extended fertility parameters is proposed to cover the endpoints repeated dose toxicity with the test substance 3,5,5 trimethylhexyl 3,5,5-trimethylhexanoate (RN CAS: 59219-71 -5).

Justification for classification or non-classification

Under the experimental conditions of the key study (Manciaux, 2001, GLP, OECD Guideline 407 Method, Klimisch 2), 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 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. The results suggested that the test item ester was hydrolysis after oral administration and the effects observed are linked to the metabolism of the fatty acid generated.

Considering the dose effect, the severity of the effects which refers to known mechanism of fatty acid.

According to the CLP criteria, the substance should not be classified with a STOT.