3,7,11-trimethyldodeca-1,6,10-trien-3-ol,mixed isomers

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

combined repeated dose and reproduction / developmental screening test with dosing duration >28 days

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,7,11-trimethyldodeca-1,6,10-trien-3-ol,mixed isomers

EC Number:

230-597-5

EC Name:

3,7,11-trimethyldodeca-1,6,10-trien-3-ol,mixed isomers

Cas Number:

7212-44-4

Molecular formula:

C15H26O

IUPAC Name:

3,7,11-trimethyldodeca-1,6,10-trien-3-ol

Details on test material:

- Name of test material (as cited in study report): Nerolidol
- Physical state and colour: Liquid/colorless, clear
- Analytical purity: sum of isomers: 99.1 %
- Lot/batch No.: 01940329U0
- Stability under test conditions: Expiry date Oct 2011
- Storage condition of test material: Room temperature; avoid temperatures >50 °C

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wistar rats Crl:WI(Han) were supplied Charles River Laboratories, Research Models and Services, Germany GmbH.
- Age at study initiation: 11-12 weeks.
- Weight at study initiation: The body weight of the males was in the range of 111.0-141.1 g (group mean: 130.4 g) and the body weight of the females was in the range of 102.4 - 123.7 g (group mean: 114.6 g).

- HOUSING: The rats were single housed in Makrolon cages type M III on type Lignocel PS 14 fibres, dustfree bedding and wooden gnawing blocks (Typ NGM E-022) as enrichment. Motor activity measurements were conducted in clean polycarbonate cages. The animals were housed in a fully air-conditioned room.
- DIET: Ground Kliba maintenance diet mouse/rat “GLP”; ad libitum.
- WATER: Drinking water from bottles ad libitum
- Acclimation period: 7 days (including randomization).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70% relative humidity
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 2009-10-19 (first administration, males and females) To: 2009-11-24 (sacrifice of parental males after withdrawal of food for about 16 - 20 hours) and 2009-12-15 (sacrifice of parental females after withdrawal of food for about 16 - 20 hours).

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION:
The test substance was weighed and thoroughly mixed with a small amount of food. Corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentration. Mixing was carried out for 10 minutes in a Ruberg (EM 100) laboratory mixer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis confirmed the correctness of the concentrations. The recovery rates were within a range of 91.0 - 97.2% of the target concentrations.

Duration of treatment / exposure:

Males 37 days, females 58 days

Frequency of treatment:

The test substance was administered daily via the diet.

Doses / concentrations

Remarks:

Doses / Concentrations:
0; 1500; 4000; 12000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10 rats per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Doses are approximatelx equivalent to a test substance intake of 100, 300 and 1000 mg/kg body weight.

- Rationale for animal assignment: Before the start of the administration period, male and female rats were allocated to the test groups according to weight. The list of randomization instructions was compiled by a computer.

Positive control:

No positive control

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Home cage observation: The animals were examined for mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occured, the animals were examined several times daily. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter.
The following parameters were examined:
1. behavior during "handling"
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalus
15. feces (appearance/consistency)
16. urine
17. pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were determind weekly. Exceptions:
• During the mating period, the females will be weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter will be weighed on the day of parturition (PND 1) and on PND 4.

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, except for the mating period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was observed daily by visual inspection of the water bottles.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the administration period, in the morning.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: All surviving animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Towards the end of the administration period.
- Animals fasted: Yes
- How many animals: All surviving animals

URINALYSIS: Yes
- Time schedule for collection of urine: Towards the end of the administration period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIORAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the administration period.
- Dose groups that were examined: five males and females per dose group were examined.
- Sensory activity, grip strength, motor activity, and reflex tests as part of Functional observational battery (FOB):

The animals were removed from the open field and subjected to following sensorimotor or reflex tests:
1. approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. winking reflex
6. pinna reflex
7. audition ("startle response")
8. olfaction
9. examination of catalepsy ("descending from box")
10. coordination of movements ("righting response")
11. behaviour during "handling"
12. vocalization
13. pain perception ("tail pinch")
14. grip strength of forelimbs
15. grip strength of hindlimbs
16. landing foot-splay test

Motor activity assessment:
Motor activity was measured on the same day as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages. The animals were put into the cages in a randomized order. The measurements started at about 14:00 h and the number of beam interrupts was counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.

Sacrifice and pathology:

NECROPSY
All F0 parental animals were sacrificed by decapitation using Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.


ORGAN WEIGHTS
The following weights were determined in all parental animals sacrificed on schedule:

1. Adrenal glands
2. Anesthetized animals
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland
11. Prostate
12. Testes
13. Seminal vesicles including coagulation glands
14. Spleen
15. Thymus
16. Thyroid glands (with parathyroid glands)
17. Uterus

ORGAN / TISSUE FIXATION OF PARENTAL ANIMALS
The following organs or tissues of parental animals were fixed in 4% buffered formaldehyde solution:

1. adrenal glands
2. all gross lesions
3. aorta
4. brain
5. bone marrow (femur)
6. cecum
7. coagulation glands
8. colon
9. duodenum
10. eyes with optic nerve
11. esophagus
12. female mammary gland
13. femur with knee joint
14. heart
15. ileum
16. jejunum (with Peyer’s patches)
17. kidneys
18. larynx
19. liver
20. lungs
21. lymph nodes (axillary and mesenteric)
22. nose (nasal cavity)
23. oviducts
24. pancreas
25. pharynx
26. pituitary gland
27. prostate gland
28. rectum
29. salivary glands (glandula mandibularis and glandula sublingualis)
30. sciatic nerve
31. seminal vesicles
32. skeletal muscle
33. skin
34. spinal cord (cervical, thoracic and lumbar cord)
35. spleen
36. sternum with marrow
37. stomach (forestomach and glandular stomach)
38. trachea
39. thymus
40. thyroid glands/parathyroid glands
41. urinary bladder
42. uterus*
43. vagina

Testes, epididymides and ovaries of animals that were killed as scheduled were fixed in modified Davidson’s solution.

HISTOPATHOLOGY OF PARENTAL ANIMALS
After the organs were fixed, histotechnical processing and examination by light microscopy was performed according to table 1:

A correlation between gross lesions and histopathological findings was performed.

Other examinations:

See Chapter 7.8

Statistics:

(1) Statistics of clinical examinations
- Parameters examined: Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of pups delivered per litter
Statistical test: The DUNNET test (two sided)
- Parameters examined: Feces, rearing, grip strength length hindlimbs, grip strength length hindlimbs, foot-splay test, motor activity
Statistical test: KRUSKAL-WALLIS test (two sided). Post test: Wilcoxon-test.
(2) Statistics of reproductive parameters
- Parameters examined: Number of live and dead pups and different indices (e.g. mating index, fertility index, gestation index) and number of litters with necropsy findings in pups
Statistical test: FISHER's exact test
- Parameters examined: Proportion of pups with necropsy findings per litter
Statistical test: WILCOXON test (one-sided)

(2) Statistics of clinical pathology
- Parameters examined: Clinical pathology parameters, except differential blood count
Statistical test: KRUSKAL-WALLIS test (two-sided). Post test: MANN-WHITNEY U test (two sided).
- Parameters examined: Urinalysis, except volume, colour, turbidity and specific gravity
Statistical test: FISHER's exact test

(3) Statistics of pathology
- Parameters examined: Weight parameters
Statistical test: KRUSKAL-WALLIS test (two-sided). Post test: WILCOXON test.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
12000/4000/1500 ppm:
- No mortality, no test substance related findings.

BODY WEIGHT AND FOOD CONSUMPTION
12000 ppm:
- Statistically significantly reduced food consumption in males, week 0 – 2, between 10% and 18% below controls.
- Statistically significantly reduced food consumption in females, week 0 – 1, 25% below controls.
- Statistically significantly reduced food consumption in females, entire gestation, average 20% below controls.
- Statistically significantly reduced food consumption in females, postnatal days 1 - 4, 32% below controls.

- Statistically significantly reduced mean body weight gain in males, week 0 – 1, 45% below controls.
- Statistically significantly reduced mean body weight in females, gestation, final weight 12% below controls.
- Statistically significantly reduced mean body weight in females, lactation, final weight 13% below controls.
- Statistically significantly reduced mean body weight gain in females, week 0 – 1, 73% below controls.
- Statistically significantly reduced mean body weight gain in females, gestation, average 37% below controls.
- Reduced mean body weight gain in females, lactation, 40% below controls.

TEST SUBSTANCE INTAKE
Corresponds to about 104, 270 and 755 mg/kg bw/d in males;
105, 279, 705 mg/kg bw/d in non-pregnant females;
120, 340 and 824 mg/kg bw/d in pregnant females;
193, 468, 1194 mg/kg bw/d in lactating females

DCO, FOB AND MOTOR ACTIVITY
12000/4000/1500 ppm:
- No test substance related findings.


CLINICAL PATHOLOGY
12000 ppm:
- Increased GGT values in rats of both sexes
- Reduced prothrombin time in females
- Decreased glucose levels in males
- Increased calcium levels in females

4000/1500 ppm:
No treatment-related, adverse effects

ORGAN WEIGHTS

12000 ppm:
- statistically significant absolute and relative increased liver weights in males (120% of control)
- statistically significantly decreased terminal body weight in females (95% of control)
- statistically significant absolute and relative increased liver weights in females (149% of control)

4000 ppm:
- statistically significant absolute and relative increased liver weights in females (120% of control)

GROSS PATHOLOGY
12000/4000/1500 ppm:
- no significant gross lesions

HISTOPATHOLOGY
12000 ppm:
- minimal or slight central hepatocellular hypertrophy and central fatty change in females

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 500 ppm

Sex:

female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

4 000 ppm

Sex:

male

Basis for effect level:

other: Based on reduction in food consumption and body weights, clinical pathological changes and increased liver weights. Dose corresponds to about 270 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:

not specified