4-(tricyclo[5.2.1.02,6]dec-8-ylidene)butyraldehyde

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 22 Aug to 24 Oct 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)

Version / remarks:

September 2009

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Test material

Test material information

Constituent 1

Reference substance name:

4-(tricyclo[5.2.1.02,6]dec-8-ylidene)butyraldehyde

EC Number:

250-078-7

EC Name:

4-(tricyclo[5.2.1.02,6]dec-8-ylidene)butyraldehyde

Cas Number:

30168-23-1

Molecular formula:

C14H20O

IUPAC Name:

4-[(8Z)-tricyclo[5.2.1.0^{2,6}]decan-8-ylidene]butanal

Test material form:

liquid

Specific details on test material used for the study:

Appearance: Colourless to pale yellow liquid
Batch: VE00471707
Test item storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 30 November 2017 (expiry date)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
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 9-11 weeks old.
Weight at the Initiation of Dosing: Males: 252 to 325 g. Females: 172 to 199 g.

Justification for Test System and Number of Animals
The Wistar Han rat was chosen as the animal model for this study as it is a rodent species
accepted by regulatory agencies for toxicity testing. The total number of animals to be used in
this study was considered to be the minimum required to properly characterize the effects of
the test item. This study was designed such that it did not require an unnecessary number of
animals to accomplish its objectives.
This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer
and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on
Animal Experimentation (February 1997).

Animal Identification
At study assignment, each animal was identified using a tattoo on the hind leg.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at
least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
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 50 to 71% (see deviations in Appendix 2). 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.
4.6.5.6. 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: aerosol

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 o84%, (the particle size where 84% of the particle mass was borne
by particles smaller than the o84% was read from the graph. The geometric standard
deviation (gsd) was calculated as o84% / MMAD.

Details on inhalation 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 to
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.
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 15 day post exposure observation period

Concentrations:

5mg/L and 1mg/L

No. of animals per sex per dose:

2 groups of 3 males and 3 females

Control animals:

no

Details on study design:

In-life Procedures, Observations, and Measurements
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. All macroscopic abnormalities found at necropsy were
preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).
For abnormalities found in the respiratory tract, the entire respiratory tract was preserved. No
histopathology was performed. On completion of the necropsy, the animals’ carcasses were
disposed of and no tissues were retained.

Statistics:

All results presented in the tables of the report are calculated using values as per the raw data
rounding procedure and may not be exactly reproduced from the individual data presented.
The LC50 value of the test item will be ranked within the following ranges: >0 - ≤0.05,
>0.05 - ≤0.5, >0.5 - ≤1, >1 - ≤5 or as exceeding 5 mg/L or the maximum achievable
concentration. No statistical analysis will be 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 and one male and all females were sacrificed
in moribund condition on Days 8 or 9. The remaining male survived until the end of the
observation period. At 1 mg/L, no mortality occurred.

Clinical signs:

other: At 5 mg/L, slow breathing was seen during exposure (not presented in the table). After exposure, lethargy, hunched posture, uncoordinated movements, labored respiration, rales, alopecia, piloerection, scales (shoulder left), chromodacryorrhoea (neck and/o

Body weight:

Body weight loss was noted for the surviving animal at 5 mg/L.
At 1 mg/L, body weight loss was noted for all animals during the first week. These animals
regained weight during the second week.

Gross pathology:

Macroscopic post mortem examination revealed abnormalities of the lungs (many dark red
foci or not collapsed) for animals that died or were sacrificed in moribund condition during
the study (5 mg/L). Macroscopic examination of the surviving animals at termination did not
reveal any abnormalities.

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The inhalation LC50, 4h value of Dupical 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 Dupical 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 guidelines described in:

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

2009.

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

three male and three female 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 5.1 ± 0.1 mg/L. The nominal

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

5.8 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 88%.

At 1 mg/L, the time-weighted mean actual concentration was 1.05 ± 0.03 mg/L. The nominal

concentration was 1.7 mg/L and the generation efficiency was 63%.

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 these variations were taken into

account resulting in an actual reflection of the mean exposure concentration over time.

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

were determined twice during each exposure period. At 5 mg/L, the MMAD was 3.3 μm (gsd

1.9) and 3.5 μm (gsd 2.1). At 1 mg/L, the MMAD was 3.1 μm (gsd 1.9) and

3.0 μm (gsd 1.9).

At 5 mg/L, one male was found dead on Day 2 and one male and all females were sacrificed

in moribund condition on Days 8 or 9. The remaining male survived until the end of the

observation period. At 1 mg/L, no mortality occurred.

At 5 mg/L, slow breathing was seen during exposure. After exposure, lethargy, hunched

posture, uncoordinated movements, labored respiration, rales, alopecia, piloerection, scales

(shoulder left), chromodacryorrhoea (neck and/or nose), lean appearance and ptosis were seen

for the animals. The surviving animals had not completely recovered from these symptoms at

the end of the observation period. At 1 mg/L, no clinical signs were seen during exposure.

After exposure, lethargy, hunched posture and shallow respiration were seen for the animals.

The animals had recovered from the clinical signs by Day 2 (males) or 3 (females).

Body weight loss was noted for the surviving animal at 5 mg/L. At 1 mg/L, Body weight loss

was noted for all animals during the first week. These animals regained weight during the

second week.

Macroscopic post mortem examination revealed abnormalities of the lungs (many dark red

foci or not collapsed) for animals that died or were sacrificed in moribund condition during

the study (5 mg/L). Macroscopic examination of the surviving animals at termination did not

reveal any abnormalities.

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

of >1 - ≤5 mg/L.

Therefore Dupical is classified as ATI category 4, harmful if inhaled (H332).