3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Aug - 3 Sep 2001

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; no detailed clinical observations were made; no neurobehavioural tests were performed; prostate and epididymides were not weighed.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted Oct 2008

Deviations:

yes

Remarks:

no detailed clinical observations made; no neurobehavioural tests performed; prostate, testes and epididymides not weighed

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate

EC Number:

261-665-2

EC Name:

3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate

Cas Number:

59219-71-5

Molecular formula:

C18H36O2

IUPAC Name:

3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate

Details on test material:

- Name of test material (as cited in study report): isononyl isononanoate
- Physical state: colourless liquid
- Analytical purity: no data
- Storage condition of test material: at room temperature

Test animals

Species:

rat

Strain:

other: Sprague-Dawley Crl CD(SD) IGS BR, COBS-VAF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France, Saint Aubin-lès-Elbeuf, France
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 204 g (males), 169 g (females)
- Fasting period before study: no
- Housing: the animals were housed 2 per cage, according to sex and dose group, in suspended wire-mesh cages (43.0 cm x 21.5 cm x 18.0 cm). A metallic tray containing autoclaved sawdust (SICSA, Alfortville, France) was placed under each cage and the sawdust changed once per week.
- Diet: A04 C pelleted maintenance diet, batch No. 90528 (U.A.R., Villemoisson-sur-Orge, France), ad libitum
- Water: tap water filtered using a 0.22 micron filter, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 04 Aug 2001 To: 2-3 Sep 2001

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: the dosing solutions were made daily by mixing the test substance with the vehicle to the required concentration of 20, 60 and 200 mg/mL and homogenising the solutions using a magnetic stirrer. The solutions were stirred continously during the dosing procedure. Doses were adjusted acording to the most recently recorded body weight of each animal.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 107H1649 (Sigma, Saint-Quentin-Fallavier, France)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

29-30 days

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and signs of morbidity, at least once daily for clinical signs

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 (prior to administration), 8, 15, 22 and 29 (prior to sacrifice)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, based on intake recorded once per week
- 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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 of the study period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: yes, overnight (at least 14 hours)
- How many animals: all the animals in the control group and treatment groups
- Parameters examined: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes (platelets), total leucocytes, differential leucocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes), reticulocytes, prothrombin time, activated partial thromboplastin time, fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28 of the study period
- Animals fasted: yes, overnight (at least 14 hours)
- How many animals: all the animals in the control group and treatment groups
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total protein, albumin, albumin/globulin ratio, cholesterol, triglycerides, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase

URINALYSIS: Yes
- Time schedule for collection of urine: Day 28 of the study period
- Metabolism cages used for collection of urine: yes
- Animals fasted: yes, overnight (at least 14 hours)
- Parameters examined: volume, pH, specific gravity, proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, sediment (microscopic cytology determining leucoytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals, cells), appearance, colour

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Gross pathology was performed in all animals, including 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 associated organs and tissues; the neck with associated organs and tissues. The absolute and relative weight of the following organs was determined in all animals: testes, heart, liver, spleen, adrenal glands, kidney, ovaries, thymus, thyroids with parathyroids. The organs were fixed in 10% neutral formaline.

HISTOPATHOLOGY: Yes. In all animals the following organs were preserved in 10% buffered formalin (except for the eyes and Harderian glands that were fixed in Davidson's fixative, and the testes and epididymides that were preserved in Bouin's fluid), embedded in paraffin wax, sectioned and stained with hematoxylin-eosin: adrenals, aorta, brain, caecum, colon, duodenum, epididymides, oesophagus, eyes with Harderian glands, femoral bone with articulation, heart, ileum, jejenum, kidneys, liver, lungs with bronchi, mandibular lymph nodes, mesenterial lymph nodes, mammary gland, ovaries, pancreas, prostate, pituitary gland, rectum, sublingual and submaxillary salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal chord (cervical, thoracic, lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids with parathyroids, tongue, trachea, urinary bladder, uterus with horns and cervix, and vagina. The above-mentioned organs and tissues were examined microscopically in animals of the control group and high-dose group, and animals that died or were killed prematurely. In addition, microscopic examination was performed on all macroscopic lesions and the target organs (liver and kidney) of the animals in the low-and mid-dose groups sacrificed at the scheduled time.

Statistics:

Body weight, food consumption, hematology, blood biochemistry, urinalysis and organ weight data were analysed for statistical significance. The tests were applied according to the flow chart attached in 'Attached background material'.

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):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

When compared to the mean control values, a higher mean monocyte level was noted with doserelationship
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 histor
ical data.
-a higher mean inorganic phosphorus level in females given 100 or 300 mg/kg /day and in males an
d 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

Description (incidence and severity):

The higher kidneys weights observed in the males were considered treatment related, could b
e 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.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/day: increased kidney weight (males, non-adverse)

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 m
g/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 d
ose 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 con
trol females, in 5 males of the 100 mg/kg/day group, 7 males of the intermediate dose group, 9 male
s 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. Sl
ight 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 consi
dered 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 gro
up) ,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

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

gross pathology

histopathology: non-neoplastic

mortality

2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

histopathology: neoplastic

mortality

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: Body weight

	Group (mg/kg bw/day)
	Males				Females
Day	Control	100	300	1000	Control	100	300	1000
1	204 ± 11.0	203 ± 11.1	206 ± 10.6	204 ± 9.3	168 ± 9.3	169 ± 8.3	167 ± 6.3	171 ± 8.9
8	263 ± 15.2	259 ± 14.8	261 ± 11.6	243 ± 26.2*	194 ± 10.7	195 ± 10.9	190 ± 8.9	173 ± 16.7**
15	308 ± 19.9	307 ± 20.5	305 ± 14.7	289 ± 30.2	216 ± 13.9	221 ± 17.2	212 ± 9.9	198 ± 28.3
22	337 ± 23.5	332 ± 31.0	329 ±16.5	317 ± 27.3	233 ± 17.5	241 ± 21.2	231 ± 8.6	223 ± 25.8
29	355 ± 26.1	346 ± 36.2	337 ± 19.6	310 ± 29.3**	240 ± 16.8	250 ± 23.5	235 ± 9.3	214 ± 36.6

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 2: Body weight Day 1 - 28

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Total body weight gain (g)	+151	+143	+131	+106	+72	+81	+68	+43
Body weight change Day 1 - 28 (relative)	x 1.7	x 1.7	x 1.6	x 1.5	x 1.4	x 1.5	x 1.4	x 1.3
Difference from control (%)	-	-3	-5	-13	-	-4	-2	-11

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 3: Selected haematology results

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Monocytes (G/L)	0.28 ± 0.094	0.32 ± 0.155	0.46 ± 0.332	0.47 ± 0.258	0.17 ± 0.077	0.19 ± 0.077	0.25 ± 0.104	0.36 ± 0.095**
WBC	10.78 ± 1.97	8.45 ± 2.04	9.84 ± 3.75	10.36 ± 2.07	6.28 ± 1.26	6.74 ± 1.63	7.57 ± 2.30	6.00 ± 1.19
APTT	23.2 ± 3.70	24.7 ± 3.67	24.5 ± 5.80	17.9 ± 4.51*	16.1 ± 2.71	15.4 ± 1.32	14.1 ± 1.43	15.5 ± 3.15

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 4: Selected clinical chemistry results

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Calcium (mmol/L)	2.66 ± 0.05	2.78 ± 0.07**	2.75 ± 0.07*	2.74 ± 0.08*	2.77 ± 0.06	2.62 ± 0.06*	2.67 ± 0.09**	2.62 ± 0.08**
Inorganic phosphate (mmol/L)	2.69 ± 0.197	2.69 ± 0.157	2.73 ± 0.100	2.94 ± 0.192**	1.98 ± 0.255	2.30 ± 0.279*	2.41 ± 0.123**	2.58 ± 0.223**
Glucose (mmol/L)	7.17 ± 0.592	5.85 ± 0.436**	5.50 ± 0.190**	5.49 ± 0.684**	7.08 ± 0.806	6.13 ± 0.489*	5.70 ± 0.885**	5.81 ± 1.193*
Urea (mmol/L)	3.5 ± 0.59	4.1 ± 0.59	4.5 ± 0.63**	4.3 ± 0.48*	5.1 ± 0.48	6.0 ± 1.10	6.5 ± 1.60*	6.6 ± 1.02*
ALP (IU/L)	313 ± 35.6	312 ± 62.6	279 ± 51.4	338 ± 111.8	190 ± 40.4	244 ± 73.5	316 ± 118.7**	364 ± 158.9**
ASAT (IU/L)	57 ± 6.3	56 ± 7.2	69 ± 13.9	86 ± 29.1**	59 ± 15.2	50 ± 18.9	75 ± 17.9	120 ± 41.1**
ALAT (IU/L)	14 ± 2.2	18 ± 4.5	21 ± 8.0	42 ± 25.9**	14 ± 5.3	24 ± 10.1	35 ± 12.3**	48 ± 15.8**

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

Table 5: Urinary volume results

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Volume (mL)	14 ± 11.2	14 ± 7.7	19 ± 7.8	28 ± 12.0*	11 ± 4.2	23 ± 9.0*	17 ± 14.1	27 ± 13.6*

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

Table 6: Selected organ weights

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Kidney, absolute (g)	2.62 ± 0.249	3.09 ± 0.320*	3.00 ± 0.287	2.98 ± 0.536	1.79 ± 0.168	2.01 ± 0.189*	1.85 ± 0.95	1.91 ± 0.255
Kidney, relative (%)	0.786 ± 0.047	0.965 ± 0.097**	0.964 ± 0.084**	1.03 ± 0.098**	0.801 ± 0.061	0.882 ± 0.062**	0.877 ± 0.043*	0.990 ± 0.052**
Liver, absolute (g)	10.9 ± 1.84	11.26 ± 1.47	13.02 ± 0.820*	16.20 ± 3.60**	7.32 ± 0.750	9.69 ± 1.42*	11.35 ± 1.46**	13.60 ± 4.48**
Liver, relative (%)	3.27 ± 0.385	3.49 ± 0.142	4.19 ± 0.330**	5.62 ± 1.01**	3.27 ± 0.247	4.23 ± 0.309	5.38 ± 0.559**	7.02 ± 2.22**
Spleen, absolute (g)	0.651 ± 0.127	0.626 ± 0.139	0.580 ± 0.117	0.502 ± 0.102*	0.534 ± 0.097	0.538 ± 0.102	0.427 ± 0.060*	0.333 ± 0.115*
Spleen, relative (%)	0.195 ± 0.032	0.194 ± 0.030	0.185 ± 0.029	0.174 ± 0.023	0.239 ± 0.042	0.235 ± 0.033	0.203 ± 0.028	0.170 ± 0.042**
Thymus, absolute (g)	0.469 ± 0.152	0.452 ± 0.104	0.426 ± 0.075	0.360 ± 0.071	0.412 ± 0.058	0.411 ± 0.058	0.328 ± 0.058*	0.260 ± 0.132**
Thymus, relative (%)	0.140 ± 0.042	0.140 ± 0.024	0.137 ± 0.022	0.127 ± 0.030	0.184 ± 0.021	0.180 ± 0.013	0.155 ± 0.021*	0.130 ± 0.050
Ovaries, absolute (g)	-	-	-	-	0.121 ± 0.017	0.129 ± 0.017	0.117 ± 0.012	0.099 ± 0.016*
Ovaries, relative (%)	-	-	-	-	0.054 ± 0.007	0.056 ± 0.006	0.056 ± 0.006	0.051 ± 0.007
Testes, absolute (g)	3.25 ± 0.258	3.15 ± 0.335	3.18 ± 0.193	3.11 ± 0.328	-	-	-	-
Testes, relative (%)	0.978 ± 0.089	0.985 ± 0.107	1.02 ± 0.071	1.09 ± 0.094*	-	-	-	-

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 7: Gross pathology results (incl. deaths/interim sacrifices)

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Liver, accentuated lobular pattern	0/10	4/10	4/10	8/10	0/10	7/10	9/10	3/10
Liver, enlarged	0/10	1/10	1/10	6/10	0/10	0/10	1/10	5/10
Liver, paleness	0/10	0/10	0/10	2/10	0/10	4/10	3/10	7/10
Kidney, grey/green colour	0/10	1/10	3/10	5/10	0/10	1/10	2/10	1/10
Spleen, reduced in size	0/10	0/10	0/10	1/10	0/10	0/10	1/10	4/10
Thymus, reduced in size	0/10	0/10	0/10	0/10	0/10	0/10	0/10	2/10

 

Table 8: Histopathology results

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Liver, moderate to severe steatosis	0/10	5/10	7/10	8/10	5/10	10/10	10/10	10/10
Liver, slight to severe hepatocellular hypertrophy	0/10	0/10	9/10	9/10	0/10	0/10	10/10	10/10
Kidney, acidophilic globules in cortical tubular epithelium, minimal to slight	0/10	10/10	10/10	10/10	0/10	0/10	0/10	0/10
Kidney, vacuolated cortical tubular epithelium, minimal to moderate	0/10	0/10	0/10	0/10	0/10	0/10	2/10	6/10
Spleen, contracted	0/10	0/10	0/10	0/10	0/10	0/10	0/10	5/10
Thymus, lymphoid depletion	0/10	0/10	0/10	0/10	0/10	0/10	0/10	7/10

 

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 acti
vities 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 3,5,5 trimethyl 3,5,5 trimethylhexanoate 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.