Dioctyl ether

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 20 - Sp 18, 2008 (main study) or Oct 30, 2008 (recovery animals)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dioctyl ether

EC Number:

211-112-6

EC Name:

Dioctyl ether

Cas Number:

629-82-3

Molecular formula:

C16H34O

IUPAC Name:

1-(octyloxy)octane

Test material form:

liquid

Details on test material:

- State of aggregation: Colorless, clear liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): C-SAT 080029
- Physical state: colourless liquid
- Analytical purity: 99.1%
- Composition of test material, percentage of components: dioctylether: 99.1%
- Purity test date: February 27, 2008
- Lot/batch No.: CE72530027
- Expiration date of the lot/batch: September 09, 2009
- Stability under test conditions: September 09, 2009
- Storage condition of test material: at room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: at start of adaptation: males: 26 days, females: 27 days
- Weight at study initiation: at start of adaptation: males: 72.9-84.0 g; females: 70.9-82.9 g
- Fasting period before study: no
- Housing: singly
- Diet (e.g. ad libitum): conventional laboratory diet ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 9 adaptation days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C (maximum range)
- Humidity (%): 55% +/- 15% (maximum range)
- Air changes (per hr): 12 - 18 times
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: June 20, 2008 To: September 18, 2008 (main study animals) or October 30, 2008 (recovery animals)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item-vehicle mixtures were freshly prepared every day. The test item was suspended in the vehicle (sunflower oil) to the appropriate
concentrations and was administered orally by gavage at a constant volume of 5 mL/kg b.w. daily for 90 days.


DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:


VEHICLE
- Justification for use and choice of vehicle (if other than water): most suitable vehicle
- Concentration in vehicle: 0.02, 0.06, 0.2 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg b.w.day
- Lot/batch no. (if required): product no. 88921, LOT: 1379622
- Purity: 100%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The determination of the concentration of C-SAT 080029 in the vehicle sunflower oil was performed using a gas chromatography (GC) method with FID detection.
The concentration of C-SAT 080029 in the mixture was quantified using a calibration curve calculated from defined peak areas of the test item. The
analytical method used was developed and validated by LPT for linearity of the calibration curve, accuracy, precision, stability, specificity and
sensitivity.
The measured concentrations ranged from 100.43% to 109.72% of the theoretical value and were well within the admissible limits.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily, 7 days each week for 90 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

main study: 10 animals / sex / group in groups 1 to 4; recovery period: 5 animals / sex / group in groups 1 and 4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 7-day dose-range-finding study (LPT Study No. 22830)
- Rationale for animal assignment (if not random): rats were selected because of their proven suitability in toxicology studies and to comply with
regulatory requirements for testing in a rodent animal species
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: 6-week recovery period
- Section schedule rationale (if not random):

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: regularly throughout the working day from 7:30 a.m. to 4:30 p.m., on Saturdays and Sundays from 8:00 a.m. to 12:00 p.m. with a
final check at approx. 4:00 p.m.
- Cage side observations checked: skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour
patterns

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of allocation of animals to groups, on the day of commencement of treatment and weekly thereafter
always on the same day of the week throughout the experimental period

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: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data:

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):
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to start of administration and at study termination (main study and recovery period)
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at main study termination (on the first day of dissection); at the end of the recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (overnight)
- How many animals: 10 / sex / group
- Parameters examined: haemoglobin content (HGB), erythrocytes (RBC), leucocytes (WBC), reticulocytes (reti), platelets (PCT), differential blood count (relative and absolute), haematocrit value (HCT), thromboplastin time (TPT), activated partial thromboplastin time (aPTT), mean corpuscular
volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at main study termination (on the first day of dissection); at the end of the recovery period
- Animals fasted: Yes (overnight)
- How many animals: 10 / sex / group
- Parameters examined: albumin, globulin, albumin/globulin ratio, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), triglycerides, urea (blood urea nitrogen), calcium, chloride, potassium, sodium, alanine aminotransferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH)

URINALYSIS: Yes
- Time schedule for collection of urine: at main study termination (on the first day of dissection); at the end of the recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight)
- Parameters examined: volume, pH, specific gravity, protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin, nitrite
microscopic examination of deposits (epithelial cells, leucocytes, erythrocytes, organisms, further constituents (i.e. sperm, casts), crystalluria)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in test week 13 approx. 1 - 2 hours after dosing and before any blood sampling
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity

OTHER: Observational screening (righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, lim rotation and auditory function)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, in all animals of all groups
HISTOPATHOLOGY: Yes, restricted to gropus 1 (vehicle control) and group 4 (high dose)

Statistics:

Students; t-test: all numerical function tests / urinalysis
Multiple t-test based on Dunnett, C.W.: body weight, food consumption, haematology, clinical biochemistry, organ weights (absolute and relative)
Exact test of R.A. Fisher: histopathology

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related clinical signs of systemic toxicity were noted in the animals treated with 100, 300 or 1000 mg/kg b.w./day. The faeces of all the control and test itemtreated animals showed a normal consistency
during the entire treatment period.

Mortality:

no mortality observed

Description (incidence):

No test item-related mortality occured.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight gain were not influenced in male and female rats treated with 100, 300 or 1000 mg/kg b.w./day.

Food efficiency:

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the food consumption for the male and female rats of all groups treated with the test item.
The visual appraisal of the drinking water consumption revealed no differences between the control and the test item-treated animals.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmological examinations of the ocular structures revealed no lesions of the eyes or the optic region.

Haematological findings:

no effects observed

Description (incidence and severity):

No test item related influence was noted.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item related influence was noted.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item related influence was noted.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The observational and functional screening in test week 13 approximately 1 to 2 hours after application did not reveal any test item-related changes at any dose level. No influence was noted on fore- and hindlimb grip strength or spontaneous motility.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The increased liver weights (relative and absolute) and kidney weights (absolute) noted in the high dosed male and female rats are considered to be a non-specific adaptive change to the high work load of the liver and kidney caused by the high dose level of 1000 mg/kg b.w./day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic inspection at necropsy did not reveal any changes in the organs and tissues of the rats treated with 100, 300 or 1000 mg/kg b.w./day.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The histopathological examination of the animals treated with 1000 mg/kg b.w./day for 90 days did not reveal any morphological lesions which are considered to be test item-related.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects at the limit dose; adaptive enlargement of liver and kidney

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: liver, kidney

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 1: Differences in organ weights to the control

Differences in organ weights to the control [%]
Organ		1000 mg/kg (group 4)
		Males	Females
Liver	Relative Absolute	+ 21 ** + 28 **	+ 14 ** + 14
Kidney (left) Kidney (right)	Absolute Absolute	+ 14 ** + 12	None None

** statistically significant at p≤0.01

Table 2: Relative Organ weights - Individual Data

Group	Animal No.	Kidney		Liver
		Left	Right
1: Control – male animals	1 2 3 4 5 6 7 8 9 10   Mean SD t	2.88 3.49 2.88 3.46 3.41 3.01 2.99 3.10 3.47 3.45   3.214 0.263 -	3.06 3.37 3.32 3.60 3.52 3.16 3.02 2.85 3.44 3.50   3.284 0.249 -	26.9 36.7 25.3 27.4 28.0 32.1 24.3 29.9 28.9 24.9   28.44 3.76 -
2: 100 mg/kg – male animals	31 32 33 34 35 36 37 38 39 40   Mean SD t	3.32 2.64 3.41 3.27 3.42 3.23 3.14 3.42 3.00 3.91   3.276 0.327 0.499	3.03 2.80 3.81 3.34 3.52 3.40 3.00 3.50 3.41 3.91   3.372 0.350 0.664	32.9 31.6 28.3 32.7 35.2 32.3 29.5 30.5 30.3 28.8   31.21 2.13 1.871
3: 300 mg/kg – male animals	51 52 53 54 55 56 57 58 59 60   Mean SD t	3.07 3.39 3.64 3.58 3.34 3.14 3.27 3.29 2.93 3.55   3.320 0.231 0.853	3.07 3.54 3.60 3.69 3.54 3.19 3.21 3.14 3.09 3.71   3.378 0.260 0.709	30.9 33.4 32.1 40.0 29.2 36.2 35.4 28.2 30.9 28.4   32.47 3.81 2.722
4: 1000 mg/kg – male animals	71 72 73 74 75 76 77 78 79 80   Mean SD t	3.20 3.48 3.21 3.61 3.41 3.71 3.27 3.15 3.48 4.06   3.458 0.281 1.964	3.39 3.42 3.17 3.64 3.34 3.51 3.31 3.05 3.63 4.19   3.465 0.315 1.365	34.9 30.4 36.5 31.7 41.0 36.3 31.7 36.6 31.8 33.0   34.39 3.27 4.019**
1: Control – female animals	16 17 18 19 20 21 22 23 24 25   Mean SD t	3.36 3.20 3.61 3.26 3.32 3.26 3.39 3.36 3.55 3.54   3.385 0.139 -	3.78 3.29 3.57 3.30 3.16 3.52 3.57 3.63 3.29 3.66   3.477 0.203 -	28.1 32.4 29.0 34.6 32.0 32.2 32.0 35.0 29.9 32.5   31.77 2.22 -
2: 100 mg/kg – female animals	41 42 43 44 45 46 47 48 49 50   Mean SD t	3.46 3.45 3.06 0.30 4.01 3.44 3.87 3.62 3.34 3.34   3.489 0.279 0.806	3.28 3.80 3.19 3.34 3.78 3.55 3.70 3.95 3.39 3.54   3.552 0.252 0.596	27.4 33.6 30.6 32.5 32.3 32.8 34.1 37.5 33.0 39.6   33.34 3.37 1.279
3: 300 mg/kg – female animals	61 62 63 64 65 66 67 68 69 70   Mean SD t	3.49 3.94 2.96 2.92 3.17 3.52 3.21 3.23 3.86 3.71   3.401 0.360 0.124	3.55 3.90 3.00 3.38 3.41 3.52 3.55 3.27 3.73 3.66   3.497 0.251 0.159	32.8 38.5 36.8 31.0 32.5 31.6 36.8 34.6 32.5 33.5   34.06 2.52 1.866
4: 1000 mg/kg – female animals	86 87 88 89 90 91 92 93 94 95   Mean SD t	3.52 4.07 3.87 3.67 4.18 3.330 3.25 3.45 3.56 3.31   3.618 0.326 1.805	3.75 4.53 3.87 3.71 4.10 3.39 3.45 3.37 3.72 3.26   3.715 0.386 1.892	36.0 39.7 34.4 33.5 35.8 32.5 38.9 39.1 32.9 38.3   36.11 2.74 3.536**
1: Control – male animals (Recovery)	11 12 13 14 15   Mean SD t	3.64 3.61 2.87 3.13 3.65   3.380 0.359 -	4.01 3.93 3.10 3.43 3.81   3.656 0.382 -	26.0 27.8 14.0 25.5 29.0   24.46 6.01 -
4: 1000 mg/kg – male animals (Recovery)	81 82 83 84 85   Mean SD t	2.74 3.31 3.40 3.47 3.38   3.260 0.296 0.576	2.78 3.41 3.66 3.58 3.50   3.386 0.351 1.163	24.7 27.5 26.1 28.9 28.8   27.20 1.80 0.976
1: Control – female animals (Recovery)	26 27 28 29 30   Mean SD t	3.96 3.41 4.02 3.54 3.68   3.722 0.263 -	4.16 3.33 4.20 3.45 3.76   3.780 0.398 -	30.4 30.6 29.8 29.3 28.0   29.62 1.04 -
4: 1000 mg/kg – female animals (Recovery)	96 97 98 99 100   Mean SD t	3.47 3.47 3.38 3.18 3.52   3.404 0.135 2.402	3.68 3.60 3.50 3.53 3.69   3.600 0.086 0.989	28.6 31.3 30.1 30.7 28.7   29.88 1.20 0.366

** p≤0.015

Table 3: Absolute Organ Weights - Individual Data

Group	Animal No.	Kidney [g]		Liver [g]
		Left	Right
1: Control – male animals	1 2 3 4 5 6 7 8 9 10   Mean SD t	1.09 1.37 1.17 1.21 1.33 1.22 1.17 1.13 1.26 1.22   1.217 0.086 -	1.16 1.32 1.35 1.26 1.37 1.28 1.18 1.04 1.25 1.24   1.245 0.098 -	10.2 14.4 10.3 9.6 10.9 13.0 9.5 10.9 10.5 8.8   10.81 1.69 -
2: 100 mg/kg – male animals	31 32 33 34 35 36 37 38 39 40   Mean SD t	1.14 1.02 1.36 1.39 1.38 1.29 1.16 1.29 1.25 1.21   1.249 0.119 0.630	1.04 1.08 1.52 1.42 1.42 1.36 1.11 1.32 1.42 1.21   1.290 0.168 0.738	11.3 12.2 11.3 13.9 14.2 12.9 10.9 11.5 12.6 8.9   11.97 1.55 1.574
3: 300 mg/kg – male animals	51 52 53 54 55 56 57 58 59 60   Mean SD t	1.21 1.33 1.62 1.30 1.34 1.26 1.22 1.33 1.27 1.35   1.323 0.115 2.085	1.21 1.39 1.60 1.34 1.42 1.28 1.20 1.27 1.34 1.41   1.346 0.118 1.656	12.2 13.1 14.3 14.5 11.7 14.5 13.2 11.4 13.4 10.8   12.91 1.34 2.850
4: 1000 mg/kg – male animals	71 72 73 74 75 76 77 78 79 80   Mean SD t	1.34 1.20 1.33 1.39 1.49 1.45 1.36 1.24 1.41 1.66   1.387 0.130 3.344**	1.42 1.18 1.31 1.40 1.46 1.37 1.38 1.20 1.47 1.71   1.390 0.150 2.377	14.6 10.5 15.1 12.2 17.9 14.2 13.2 14.4 12.9 13.5   13.85 1.95 4.125**
1: Control – female animals	16 17 18 19 20 21 22 23 24 25   Mean SD t	0.80 0.70 0.81 0.83 0.84 0.76 0.73 0.74 0.82 0.85   0.788 0.052 -	0.90 0.72 0.80 0.84 0.80 0.82 0.77 0.80 0.76 0.88   0.809 0.054 -	6.7 7.1 6.5 8.8 8.1 7.5 6.9 7.7 6.9 7.8   7.40 0.71 -
2: 100 mg/kg – female animals	41 42 43 44 45 46 47 48 49 50   Mean SD t	0.77 0.79 0.68 0.72 0.87 0.89 0.92 0.88 0.73 0.80   0.805 0.082 0.475	0.73 0.87 0.71 0.73 0.82 0.92 0.88 0.96 0.74 0.85   0.821 0.089 0.325	6.1 7.7 6.8 7.1 7.0 8.5 8.1 9.1 7.2 9.5   7.71 1.08 0.768
3: 300 mg/kg – female animals	61 62 63 64 65 66 67 68 69 70   Mean SD t	0.66 0.85 0.74 0.64 0.80 0.89 0.76 0.70 0.89 0.82   0.775 0.090 0.363	0.67 0.84 0.75 0.74 0.86 0.89 0.84 0.71 0.86 0.81   0.797 0.074 0.325	6.2 8.3 9.2 6.8 8.2 8.0 8.7 7.5 7.5 7.4   7.78 0.89 0.941
4: 1000 mg/kg – female animals	86 87 88 89 90 91 92 93 94 95   Mean SD t	0.92 0.87 0.89 0.81 0.97 0.71 0.82 0.82 0.91 0.69   0.841 0.090 1.482	0.98 0.97 0.89 0.82 0.95 0.73 0.87 0.80 0.95 0.68   0.864 0.104 1.489	9.4 8.5 7.9 7.4 8.3 7.0 9.8 9.3 8.4 8.0   8.40 0.89 2.477
1: Control – male animals (Recovery)	11 12 13 14 15   Mean SD t	1.50 1.35 1.29 1.24 1.45   1.366 0.108 -	1.65 1.47 1.39 1.36 1.51   1.476 0.114 -	10.7 10.4 6.3 10.1 11.5   9.80 2.02 -
4: 1000 mg/kg – male animals (Recovery)	81 82 83 84 85   Mean SD t	1.32 1.29 1.30 1.60 1.35   1.372 0.129 0.079	1.34 1.33 1.40 1.65 1.40   1.424 0.130 0.670	11.9 10.7 10.0 13.3 11.5   11.48 1.25 1.577
1: Control – female animals (Recovery)	26 27 28 29 30   Mean SD t	0.99 0.86 0.93 0.75 0.96   0.898 0.096 -	1.04 0.84 0.97 0.73 0.98   0.912 0.125 -	7.6 7.7 6.9 6.2 7.3   7.14 0.61 -
4: 1000 mg/kg – female animals (Recovery)	96 97 98 99 100   Mean SD t	0.85 0.81 0.83 0.83 0.81   0.826 0.017 1.656	0.90 0.84 0.86 0.92 0.85   0.874 0.034 0.655	7.0 7.3 7.4 8.0 6.6   7.26 0.52 0.335

** p≤0.015

Applicant's summary and conclusion

Conclusions:

The NOAEL was > 1000 mg/kg bw.

Executive summary:

The oral toxicity of the test article was tested by daily administration to rats, has been investigated over a period of 13 consecutive weeks.Three groups, each of 10 male and 10 female rats (strain Crl:CD), received the test substance daily by oral gavage (5 ml) at dosages of 100, 300, and 1000 mg/kg/day for a minimum of 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (sun flower oil) and acted as a control.No death occurred during the study. Daily post‑dose observations did not show any significant signs. Detailed clinical signs with neurotoxicity assessment did not show any treatment‑related effects. Neurotoxicity tests and measurements performed at the end of the treatment did not show changes attributable to the test substance. With regard to body weight, no statistically significant differences were observed between control and treated groups. No test item-related influence was observed in food consumption. No findings were seen in the ophthalmic examination performed at the end of the study. No treatment‑related changes were observed in haematological parameters. No treatment‑related changes were seen in clinical chemistry parameters. No treatment-related pathological and histopathological changes were seen. No changes were seen in urinalysis. Treatment with 1000 mg/kg bw caused an increase in the absolute and related liver and kidney weights by up to 280 %. The increase is considered to be a non-specific adaptive change to the high work load of the liver caused by a dose level of 1000 mg/kg bw. Under the present test conditions, the no-observed-adverse-effect-level (NOAEL) for the test article was above 1000 mg/kg bw for systemic changes.