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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
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
Cross-reference
Reason / purpose for cross-reference:
reference to same study

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 (incl. QA statement)
Remarks:
BASF SE, Experimentelle Toxikologie und Ökologie
Limit test:
no

Test material

Constituent 1
Chemical structure
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 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.
Details on mating procedure:
- Males/Females ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: A vaginal smear was prepared after each mating and examined for sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "day 0" and the following day "day 1" p.c. (post coitum).
- After successful mating each pregnant female was caged in Makrolon cages type M III on type Lignocel PS 14 fibres, dustfree bedding and wooden gnawing blocks (Typ NGM E-022) as enrichment. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation
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 (bw).

- 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

Parental animals: Observations and examinations:
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 1400 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.
Oestrous cyclicity (parental animals):
no estrous cyclicity assessed
Sperm parameters (parental animals):
no sperm parameters assessed
Litter observations:
PARAMETERS EXAMINED:
Status (sex, liveborn or stillborn) and number of all pups delivered from the parents were determined as soon as possible after birth. At the same time, the pups were also examined for gross-morphological changes. Pups, which died before the first determination of their status on the day of birth, were designated as stillborn pups.
Parameters: pup viability/mortality; sex ratio; pup clinical observations; pup body weight; pup organ weights; pup necroscopy observations.

Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays and public holidays. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.

Clinical signs
All live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups show particular findings, these were documented with the dam concerned.


Body weights
The pups were weighed on the day after birth (PND 1) and on PND 4. The body weight determined on PND 1 will also be used to determine runts. Those pups whose body weight is > 25% below the mean body weight of the control group (separately according to male and female pups) were defined as runts.
Postmortem examinations (parental animals):
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.
Postmortem examinations (offspring):
POSTMORTEM EXAMINATIONS OF PUPS
On PND 4, the pups were sacrificed with CO2. After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically. Pups that died or were sacrificed in a moribund state were eviscerated and examined for possible defects and/or the cause of death.
All pups without any notable findings were discarded after their macroscopic evaluation.
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.
Reproductive indices:
Male:
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:

Male mating Index (%) = number of males with confirmed mating*/number of males placed with females x 100
* defined by a female with vaginal sperm or with implants in utero

Male fertility Index (%) = number of males proving their fertility*/number of males placed with females x 100
* defined by a female with implants in utero

Female:
The mating partners, the number of mating days until vaginal sperm could be detected, and gestational status were recorded for F0 females. For the females, mating, fertility and gestation indices were calculated for FI and F2 litters according to the following formulas:

Female mating Index (%) = number of females mated* / number of females piaced with males x 100
* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = number of femals pregnant*/ number of females mated** x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation Index (%) = number of females with live pups an the day of birth/ number of females pregnant* x 100
* defined as the number of females with implants in utero
Offspring viability indices:
Viability index was calculated according to the following formula:
Viability Index (%) = number of live pups an day 4 after birth/number of live pups an the day of birth x 100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
F0 animals:
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.

REPRODUCTIVE PERFORMANCE

12000/4000/1500 ppm:
- No test substance related effects on mating, gestation and parturition
- 1 low dose and 1 high dose male did not generate F1 pups. In line, the respective females did not become pregnant resulting in fertility indices of 100%, 90%, 100% and 90% in 0, 1500 ppm, 4000 ppm and 12000 ppm respectively. No histomorphological correlate was found in males and females to explain the apparent infertilities. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.


12000 ppm:
- Slightly, non-significantly lower number of implants and, subsequently litter size

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 (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
4 000 ppm
Sex:
male
Basis for effect level:
other: General toxicity 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.
Dose descriptor:
NOAEL
Effect level:
1 500 ppm
Sex:
female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
>= 12 000 ppm
Sex:
male/female
Basis for effect level:
other: Fertility and reproductive performance based on unchanged reproductive indices. Dose corresponds to about 755 mg/kg bw/d in males and 705, 824 or 1194 mg/kg bw/d in non-pregnant, pregnant or lactating females, respectively.

Results: F1 generation

Details on results (F1)

VIABILITY

12000/4000/1500 ppm:
- No mortality

CLINICAL SIGNS
12000/4000/1500 ppm:
- No test substance-related findings

BODY WEIGHT
12000 ppm:
- Statistically significantly reduced mean pup body weight, postnatal day 4, 13% below controls.
- Statistically significantly reduced mean pup body weight gain, postnatal days 1 - 4, 38% below controls.

PUP NECROPSY
12000/4000/1500 ppm:
- No test substance-related findings

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
Developmental
Generation:
F1
Effect level:
4 000 ppm
Sex:
male/female
Basis for effect level:
other: Developmental toxicity based on growth and development of offspring, secondary to maternal toxicity. Dose corresponds to about 270 mg/kg bw/d in males and 279, 340 or 468 mg/kg bw/d in non-pregnant, pregnant or lactating females, respectively.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion