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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 08 May 2017 to 28 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(3Z,6Z)-3,7,11-trimethyldodeca-1,3,6,10-tetraene
Cas Number:
28973-99-1
Molecular formula:
C15H24
IUPAC Name:
(3Z,6Z)-3,7,11-trimethyldodeca-1,3,6,10-tetraene
Constituent 2
Chemical structure
Reference substance name:
(E)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)cyclohex-1-ene
Cas Number:
25532-79-0
Molecular formula:
C15H24
IUPAC Name:
(E)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)cyclohex-1-ene
Constituent 3
Chemical structure
Reference substance name:
(Z)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)
Cas Number:
29837-07-8
Molecular formula:
C15H24
IUPAC Name:
(Z)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)
Constituent 4
Chemical structure
Reference substance name:
(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
EC Number:
242-582-0
EC Name:
(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Cas Number:
18794-84-8
Molecular formula:
C15H24
IUPAC Name:
7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Constituent 5
Chemical structure
Reference substance name:
(E)-1-methyl-4-(6-methylhept-5-en-2-ylidene)
Cas Number:
53585-13-0
Molecular formula:
C15H24
IUPAC Name:
(E)-1-methyl-4-(6-methylhept-5-en-2-ylidene)
Constituent 6
Chemical structure
Reference substance name:
(Z)-1-methyl-4-(6-methylhept-5-en-2-ylidene)
Cas Number:
13062-00-5
Molecular formula:
C15H24
IUPAC Name:
(Z)-1-methyl-4-(6-methylhept-5-en-2-ylidene)
Constituent 7
Chemical structure
Reference substance name:
1-methyl-4-(6-methylhepta-1,5-dien-2-yl)
Cas Number:
869843-05-0
Molecular formula:
C15H24
IUPAC Name:
1-methyl-4-(6-methylhepta-1,5-dien-2-yl)
Constituent 8
Chemical structure
Reference substance name:
(3Z,6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene
Cas Number:
26560-14-5
Molecular formula:
C15H24
IUPAC Name:
(3Z,6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene
Constituent 9
Chemical structure
Reference substance name:
(Z)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Cas Number:
28973-97-9
Molecular formula:
C15H24
IUPAC Name:
(Z)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Constituent 10
Chemical structure
Reference substance name:
1-methyl-4-(6-methylhept-5-en-2-yl)cyclohexa-1,4-diene
Cas Number:
72345-84-7
Molecular formula:
C15H24
IUPAC Name:
1-methyl-4-(6-methylhept-5-en-2-yl)cyclohexa-1,4-diene
Constituent 11
Chemical structure
Reference substance name:
2,6,10-trimethyldodeca-2,6,9,11-tetraene
EC Number:
207-948-6
EC Name:
2,6,10-trimethyldodeca-2,6,9,11-tetraene
Cas Number:
502-61-4
Molecular formula:
C15H24
IUPAC Name:
3,7,11-trimethyldodeca-1,3,6,10-tetraene
Constituent 12
Chemical structure
Reference substance name:
Likely sesquiterpene hydrocarbons
Cas Number:
n/a
Molecular formula:
C15H24
IUPAC Name:
Likely sesquiterpene hydrocarbons
Test material form:
liquid
Details on test material:
UVCB substance
Specific details on test material used for the study:
Appearance: Colourless to pale yellow liquid
Batch: VE00463634
Purity/Composition: UVCB
Test item storage: At room temperature protected from light
Stable under storage conditions until: 17 October 2017 (expiry date)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species: Rat
Strain: Crl: WI(Han)
Condition: Outbred, SPF-Quality
Source: Charles River Deutschland, Sulzfeld, Germany
Number of Animals: 3 animals of both sexes per exposure group. Two
exposure groups. Females were nulliparous and nonpregnant.
Age at the Initiation of Dosing: Young adult animals, approximately 10-11 weeks old.
Weight at the Initiation of Dosing: Males: 257 to 388 g.
Females: 157 to 237 g.
At study assignment, each animal was identified using a tattoo on the hind leg.
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at
least 5 days before the commencement of dosing.
Animals were assigned to the study at the discretion of the coordinating biotechnician
according to body weights, with all animals within ± 20% of the sex mean. Animals in poor
health or at extremes of body weight range were not assigned to the study.
Before the initiation of dosing, a health inspection was performed and any assigned animal
considered unsuitable for use in the study were replaced by alternate animals obtained from
the same shipment and maintained under the same environmental conditions.
The disposition of all animals was documented in the study records.
Animals were assigned to the study at the discretion of the coordinating biotechnician
according to body weights, with all animals within ± 20% of the sex mean. Animals in poor
health or at extremes of body weight range were not assigned to the study.
Before the initiation of dosing, a health inspection was performed and any assigned animal
considered unsuitable for use in the study were replaced by alternate animals obtained from
the same shipment and maintained under the same environmental conditions.
The disposition of all animals was documented in the study records.
Husbandry
Housing
On arrival and following assignment to the study, animals were group housed (up to 5
animals of the same sex and same exposure group together) in polycarbonate cages
(Makrolon MIV type; height 18 cm.) containing sterilized sawdust as bedding material
(Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany)
equipped with water bottles. These housing conditions were maintained unless deemed
inappropriate by the Study Director and/or Clinical Veterinarian. The room(s) in which the
animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly
labeled.
Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were
maintained. The actual daily mean temperature during the study period was 20 to 21°C with
an actual daily mean relative humidity of 55 to 70%. A 12-hour light/12-hour dark cycle was
maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation)
were maintained in the animal rooms.
Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was
provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental
contaminants. Results of the analysis were provided by the supplier and are on file at the Test
Facility.
It is considered that there were no known contaminants in the feed that would interfere with
the objectives of the study.
Water
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the
Test Facility.
It is considered that there were no known contaminants in the water that would interfere with
the objectives of the study.
Animal Enrichment
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri,
Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted
by study procedures/activities.
Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations
or treatments were required.

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1 - <= 4 µm
Geometric standard deviation (GSD):
>= 1.5 - <= 3
Remark on MMAD/GSD:
The particle size distribution was characterized twice during each exposure period. The
samples were drawn with a flow of 2 L/min. from the test atmosphere through a tube mounted
in one of the free animal ports of the exposure chamber (Figure 1). The samples were
collected with an 8 stage Marple personal cascade impactor containing fiber glass filters (TE-
290-GF. Tisch Environmental, Cleves, Ohio, USA) and a fiber glass back-up filter (SEC-290-
F1, Westech, Upper Stondon, Bedfordshire, England). Amounts of test item collected were
measured gravimetrically. Subsequently the Mass Median Aerodynamic Diameter (MMAD)
and the Geometric Standard Deviation (GSD) were determined based on OECD guidance
document No 39. Graphs of the cumulative mass of test item collected (percentage of total
collected) against the cut points of the impactor stages were drawn on log-normal paper.
When drawing the graphs more weight was given to the cut points where the cumulative mass
sampled was within the range of 5 to 95%. The Mass Median Aerodynamic Diameter
(MMAD), i.e. the particle size where 50% of the particle mass was borne by particles smaller
than the MMAD and the σ84%, (the particle size where 84% of the particle mass was borne
by particles smaller than the σ84% was read from the graph. The geometric standard
deviation (gsd) was calculated as σ84% / MMAD.
Details on inhalation exposure:
Animal Husbandry on the Day of Exposure
The animals were moved to the inhalation area to in order to perform the exposure. During
exposure, there was no access to food and water. After exposure, animals were returned their
cages which were placed in a fume cupboard for a short time period to allow test item
remnants to evaporate. A sheet of filter paper was used to cover the bedding material to
prevent suffocation in case of bad health condition and to aid clinical observations. The sheet
was removed before the end of the exposure day and the surviving animals were returned to
the animal room.
Exposure Chamber
Animals were exposed to the test item via the nose only inhalation route. For this purpose the
animals were placed in polycarbonate restraining tubes, which were connected to the
exposure chamber. Animals were allowed to acclimatize for at least fifteen minutes after the
last animal has been placed.
The design of the exposure chamber is based on the directed flow nose only inhalation
chamber (Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983). The chamber consists of animal
sections with eight animal ports each. Each animal port has its own test atmosphere inlet and
exhaust outlet. The number of animal sections and number of open inlets were adapted to the
air flow in such a way that at each animal port the theoretical air flow was at least 1 L/min.
The main inlet of the test atmosphere was located at the top section and the main outlet was
located at the bottom section. The direction of the flow of the test atmosphere guaranteed a
freshly generated atmosphere for each individual animal. The placement of the individual
animals in the inhalation chamber is shown in Figure 2. All components of the exposure
chamber in contact with test item were made of stainless steel, glass, rubber or plastic. To avoid exposure of the personnel and contamination of the laboratory the exposure chamber
was placed in a fume hood, maintained at a slight negative pressure.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Remarks on duration:
Animals were retained for a 14 day post exposure observation period
Concentrations:
5mg/L and 1mg/L
No. of animals per sex per dose:
5mg/L exposure group: 3 males and 3 females
1 mg/L exposure group: 3 males and 3 females
Control animals:
no
Details on study design:
Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity
twice daily, in the morning and at the end of the working day. Animals were not removed
from cage during observation, unless necessary for identification or confirmation of possible
findings.
Clinical Observations
Observations During Exposure
Animals were checked for mortality, behavioral signs of distress and effects on respiration at
least three times during exposure.
Post Exposure Observations
Post exposure observations were performed at periodic intervals on the day of exposure (at
least two times) and once daily thereafter. The observation period was 14 days.
All the animals were examined for reaction to exposure. The onset, intensity and duration of
these signs was recorded (if appropriate), particular attention being paid to the animals for the
first hours after exposure.
Body Weights
Animals were weighed individually on Day 1 (pre exposure), 2, 4 and 8 and 15. Terminal
body weights were collected from animals found dead or euthanized moribund after Day 1.
Terminal Procedures
All moribund animals and animals surviving to the end of the observation period were
sacrificed by an intra-peritoneal injection of Euthasol® (at least 250 mg/kg). All animals
assigned to the study were subjected to necropsy and descriptions of all internal macroscopic
abnormalities were recorded. On completion of the necropsy, the animals’ carcasses were
disposed of and no tissues were retained.
Statistics:
No statistical analysis performed (the method used is not intended to allow the calculation of a precise LC50 value).

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 1 - <= 5 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
At 5 mg/L, one male was found dead on Day 2, the remaining males and females were killed
in extremis on Day 7 or 8.
At 1 mg/L, no mortality occurred.
Clinical signs:
other: At 5 mg/L, labored respiration was seen for the animals during exposure (not presented in table). After exposure, lethargy, hunched posture, labored respiration, ptosis and hypothermia were seen for the animals between Days 1 and 8, with signs worsening o
Body weight:
Overall body weight gain in the surviving males and females was within the range expected
for rats of this strain and age used in this type of study and were therefore considered not
indicative of toxicity.
Gross pathology:
At 5 mg/L macroscopic post mortem examination revealed abnormalities of the lungs
(enlarged, swollen, many grey-white foci) for the animals that were were sacrificed in
moribund condition during the study. Beginning autolysis was noted for the animal that died
during the study, which was considered not toxicologically relevant. Macroscopic
examination of the animals of 1 mg/L at termination did not reveal any abnormalities.
Other findings:
At 5 mg/L, the time-weighted mean actual concentration was 5.0 ± 0.1 mg/L. The nominal
concentration (amount of test item used divided by the volume of pressurized air used) was
5.9 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 84%.
At 1 mg/L, the time-weighted mean actual concentration was 1.0 ± 0.04 mg/L. The nominal
concentration (amount of test item used divided by the volume of pressurized air used) was
2.3 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 44%.
The concentration was measured at several time points that were equally distributed over the
exposure period, the results of which demonstrated that the item was sufficiently stable. The
variation in concentration was caused by adjustments to the generation equipment. By
calculating the time-weighted mean concentration, effects of the variations were taken into
account resulting in an actual reflection of the mean exposure concentration over time.

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The inhalation LC50,4h value of Bisabolene in Wistar rats was established to be within the range of >1 - <= 5 mg/L.
Executive summary:

The objective of this study was to assess the acute inhalation toxicity of Bisabolene in rats of

both sexes in rats following a single 4 hour nose-only exposure to one or more defined

concentrations. Animals were retained for a 14 day post exposure observation period.

The study was carried out based on the guideline described in:

• OECD Guideline 436. Acute Inhalation Toxicity- Acute Toxic Class Method, September

2009.

Bisabolene was administered as an aerosol by nose only inhalation for 4 hours to two groups

of three male and three female Wistar rats. Mortality and clinical signs were observed daily

during the observation period and body weights were determined on Days 1, 2, 4, 8 and 15.

Macroscopic examination was performed on the day of death or after terminal sacrifice (Day

15).

At 5 mg/L, the time-weighted mean actual concentration was 5.0 ± 0.1 mg/L. The nominal

concentration (amount of test item used divided by the volume of pressurized air used) was

5.9 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 84%.

At 1 mg/L, the time-weighted mean actual concentration was 1.0 ± 0.04 mg/L. The nominal

concentration (amount of test item used divided by the volume of pressurized air used) was

2.3 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 44%.

The concentration was measured at several time points that were equally distributed over the

exposure period, the results of which demonstrated that the item was sufficiently stable.

The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd)

were determined twice during the exposure period. At 5 mg/L, the MMAD was 2.3 μm (gsd

2.1) and 3.2 μm (gsd 2.2). At 1 mg/L, The MMAD was 3.0 μm (gsd 1.7) and 3.0 μm (gsd

1.9).

At 5 mg/L, one male was found dead on Day 2, the remaining males and females were killed

in extremis on Day 7 or 8. At 1 mg/L, no mortality occurred.

At 5 mg/L, labored respiration was seen for the animals during exposure. After exposure,

lethargy, hunched posture, labored respiration, ptosis and hypothermia were seen for the

animals between Days 1 and 8, with signs worsening on Day 7 or 8. At 1 mg/L, slow

breathing was seen for the animals during exposure. After exposure, fearful behavior,

lethargy, hunched posture, slow breathing and/or ptosis were seen in the males on Day 1.

No clinical signs of systemic toxicity were noted for the females.

Overall body weight gain in the surviving males and females was within the range expected

for rats of this strain and age used in this type of study and were therefore considered not

indicative of toxicity.

At 5 mg/L macroscopic post mortem examination revealed abnormalities of the lungs

(enlarged, swollen, many grey-white foci) for the animals that were were sacrificed in

moribund condition during the study. Macroscopic examination of the animals of 1 mg/L at

termination did not reveal any test item related abnormalities.

The inhalation LC50, 4h value of Bisabolene in Wistar rats was established to be within the

range of >1 - ≤5 mg/L.