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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 14, 2017 to January 10, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
GLP study conducted in compliance with OECD Guideline No. 436 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Version / remarks:
Regulation (EC) No. 260/2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
7 September 2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
(1S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
EC Number:
207-856-6
EC Name:
(1S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
Cas Number:
498-15-7
Molecular formula:
C10H16
IUPAC Name:
3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
impurity 1
Chemical structure
Reference substance name:
(1R,6S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
Cas Number:
20296-50-8
Molecular formula:
C10H16
IUPAC Name:
(1R,6S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
impurity 2
Chemical structure
Reference substance name:
(-)-pin-2(10)-ene
EC Number:
242-060-2
EC Name:
(-)-pin-2(10)-ene
Cas Number:
18172-67-3
Molecular formula:
C10H16
IUPAC Name:
(1S,5S)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
impurity 3
Chemical structure
Reference substance name:
7-methyl-3-methyleneocta-1,6-diene
EC Number:
204-622-5
EC Name:
7-methyl-3-methyleneocta-1,6-diene
Cas Number:
123-35-3
Molecular formula:
C10H16
IUPAC Name:
7-methyl-3-methyleneocta-1,6-diene
impurity 4
Chemical structure
Reference substance name:
(R)-p-mentha-1,8-diene
EC Number:
227-813-5
EC Name:
(R)-p-mentha-1,8-diene
Cas Number:
5989-27-5
Molecular formula:
C10H16
IUPAC Name:
(4R)-isopropenyl-1-methylcyclohexene
impurity 5
Chemical structure
Reference substance name:
(S)-p-mentha-1,8-diene
EC Number:
227-815-6
EC Name:
(S)-p-mentha-1,8-diene
Cas Number:
5989-54-8
Molecular formula:
C10H16
IUPAC Name:
(4S)-isopropenyl-1-methylcyclohexene
impurity 6
Chemical structure
Reference substance name:
p-mentha-1,3-diene
EC Number:
202-795-1
EC Name:
p-mentha-1,3-diene
Cas Number:
99-86-5
Molecular formula:
C10H16
IUPAC Name:
1-isopropyl-4-methylcyclohexa-1,3-diene
impurity 7
Chemical structure
Reference substance name:
3-isopropyl-(6S)-methylenecyclohexene
Cas Number:
6153-16-8
Molecular formula:
C10H16
IUPAC Name:
3-isopropyl-(6S)-methylenecyclohexene
impurity 8
Chemical structure
Reference substance name:
3-isopropyl-(6R)-methylenecyclohexene
Cas Number:
6153-17-9
Molecular formula:
C10H16
IUPAC Name:
3-isopropyl-(6R)-methylenecyclohexene
impurity 9
Chemical structure
Reference substance name:
(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
Cas Number:
19902-08-0
Molecular formula:
C10H16
IUPAC Name:
(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
Test material form:
liquid
Details on test material:
Batch No.: 177809
Purity : 83.2%
Name of test material (as cited in study report): delta-3-carene
Physical state: colourless liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 04 February 2017

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Sprague-Dawley derived, albino
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: SAGE Labs on December 6, 2017.
- Age at study initiation: Young adult (8-11 weeks); females were nulliparous and non pregnant.
- Weight at study initiation: 254 to 342 g (males) and 168 to 220 g (females).
- Fasting period before study: None.
- Housing: Housed singly in suspended stainless steel caging, with enrichments. Litter paper was placed beneath the cage and was changed at least three times per week.
- Diet: Envigo Teklad Global 16% Protein Rodent Diet #2016 was provided ad libitum throughout the study, except during designated procedures.
- Water: tap-water was freely available to each animal via water bottles.
- Acclimation period: 8 or 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 25°C
- Humidity (%): 31 - 56%
- Air changes (per hr): Twelve.
- Photoperiod: 12 h light / 12 h dark

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
> 2.18 - < 2.46 µm
Geometric standard deviation (GSD):
> 1.95 - < 2.59
Remark on MMAD/GSD:
The geometric standard deviation (gsd) will be calculated using two-cycle logarithmic probit axes.
Details on inhalation exposure:
Nose only exposure will be used since this is the most applicable method of exposure for the test model. This exposure technique is used to minimize concurrent exposure by the oral and dermal routes.

DESCRIPTION (See on the table 1)
The test substance was aerosolized as received and kept on a magnetic stirrer during aerosolization.
The design of the exposure chamber is based on the directed flow nose-only inhalation chamber (ADG Developments LTD 6.7 or 28 liter).

- Method of holding animals in test chamber
Animals will be exposed to the test item via the nose only inhalation route. For this purpose the animals were individually housed in polycarbonate holding tubes whichseal to the chamber with an "O" ring during exposure. The base unit terminates the chamber with a 0.5-inch diameter tube for discharged air.

PRE-TEST TRIALS
Prior to initiation of the main test, pre-test trials were conducted to establish generation procedures to achieve, to the extent possible, the desired chamber concentration and desired particle size distribution (1 µm - Air pressure: constant
- Compressed Generator Airflow: constant
- compressed Mixing Airflow: constant
- Total Airflow: constant
- Liquid Pump Setting: varied
- Liquid Pump Type: constant
- Tubing size: constant
- Atomization System: constant
- Clean-Out Needle and Fluid Cap: constant
- Air Cap: constant
- Vacuum Pump: constant
- Concentration Sampling Time: varied
The procedures and aerosolization equipment used in the main test were based on the results of pre-test trial No. 2 and 5. In each instance, the conditions of generation were modified to achieve the targeted chamber concentration with a desirable particle size distribution. See Table 1.

For each exposure, the exposure chamber, air supply, and equipment used to measure particle size distribution, airflow, and chamber concentration were the same as used during the respective pre-test trials.

TEST ATMOSPHERE GENERATION AND CHARACTERIZATION (See Table 2, 3 and 4)

- Test Atmosphere Generation
The test atmosphere was generated using a nebulizer and metered to the atomization nozzle through Tygon tubing, using a peristaltic pump. Filtered generator air was supplied to the spray atomization nozzle by an air compressor, and measured with a Mass Flow Controller. Additional filtered mixing air from the same air compressor, measured with a Mass Flow Controller, was introduced into the chamber to help uniformly distribute the test atmosphere by creating a vortex at the chamber inlet. Chamber airflow was monitored throughout the exposure period and recorded periodically. The exposure was conducted under slight negative pressure.

- Chamber Concentration Measurements
Gravimetric samples were withdrawn at 6 intervals from the breathing zone of the animals. Samples were collected using 37 mm glass fiber filters (Whatman TM GF/B) in a filter holder attached by 1/4 inch Tygon tubing to a vacuum pump. Filter papers were weighed before and after collection to determine the mass collected. This value was divided by the total volume of air sampled to determine the chamber concentration. Sample airflows were measured using a Mass Flow Controller.

- Particle Size Distribution Investigations
An eight-stage 1 ACFM Andersen Ambient Particle Sizing Sampler was used to access the particle size distribution of the test atmosphere. Samples were withdrawn from the breathing zone of the animals at two intervals. The filter paper collection stages were weighed before and after sampling to determine the mass collected upon each stage. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated using two-cycle logarithmic probit axes.

- Temperature and Relative Humidity of the Test Atmosphere
The temperature and relative humidity within the exposure chamber as well as the room were monitored continuously during exposure, and were measured with a temperature-humidity monitor. A humidifier was used in the room during exposure (1.05 mg/L). Temperature and relative humidity values were recorded every 15 minutes for the first hour of exposure and approximately every 15 or 30 minutes thereafter.

- Exposure period
Animals were exposed to the targetes chamber concentration for at least 4 hours. The exposure period was extended ebyond 4 hours to allow the chamber to reach equilibrium. At the end of the exposure period, the generation was terminated and the chamber was operated for at least 15 minutes further with clean air to allow the test atmosphere to fully dissipate. At the end of this period the animals were removed from the exposure tubes. Prior to being returned to their cages, excess test substance was removed from the fur of each animal by rinsing with tap-water and wiping with clean paper towels.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Remarks on duration:
See "Details on inhalation exposure"
Concentrations:
Exposure levels will be selected based on the EC and UN classification guidelines.
The starting exposure level was selected based on the available test item data:
At 5 mg/L, the mean actual exposure concentration was 5.07 ± 0.28 mg/L. The nominal concentration (amount of test item used divided by the total airflow multiplied by the total time) was 82.90 mg/L.
At 1 mg/L, the mean actual exposure concentration was 1.05 ± 0.09 mg/L. The nominal concentration (amount of test item used divided by the total airflow multiplied by the total time) was 15.62 mg/L.
No. of animals per sex per dose:
3 per sex per dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days or until death occurred.

OBSERVATIONS AND MEASUREMENTS
- Mortality/Moribundity checks : All animals were observed for mortality during the exposure period.

- Clinical observations:
The animals were examined for signs of gross toxicity, and behavioral changes upon removal from the exposure tube and at least once daily thereafter for 14 days or until death occured.
Observations included gross evaluation of skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, and coma.

Individual body weights of the animals were recorded prior to test substance exposure (initial) and again on Days 1, 3, 7, and 14 (terminal) or after death.

Five rats euthanized for humane reasons due to test substance exposure. Surviving rats were euthanized on Day 14. All animals were euthanized via CO2 inhalation. gross necropsies were performed on all decedents and euthanized animals. Tissues and organs of the thoracic and abdominal cavities were examined.

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.
Statistics:
Statistical analysis was limited to the calculation of the mean and standard deviation.

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 1.05 - <= 5.07 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
At the 5.07 mg/L dose level, one female rat was found dead at chamber removal and the remaining five animals were euthanized for humane reasons following exposure to the test atmosphere.
At the 1.05 mg/L dose level, all animals survived at the test item exposure.
See Table 6.
Clinical signs:
other: other:
Body weight:
At the 5.07 mg/L dose level, due to the mortality, no body weights changes were recorded.
At the 1.05 mg/L dose level, all animals gained body weight during the study.
See Table 5.
Gross pathology:
At the 5.07 mg/L dose level, gross necropsy of the decedents revealed discoloration of the lungs and/or distention of the intestines.
At the 1.05 mg/L dose level, no gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.
Other findings:
At the 5.07 mg/L dose level, the gravimetric chamber and nominal chamber concentrations were 5.07 mg/L and 82.90 mg/L, respectively. The average mass median aerodynamic diameter was estimated to be 2.28 µm based on graphic analysis of the particle size distribution as measured with a 1 ACFM Andersen Ambient Particle Sizing Sampler with an avergae geometric standard deviation of 2.49.
At the 1.05 mg/L dose level, the gravimetric chamber and nominal chamber concentrations were 1.05 mg/L and 15.62 mg/L, respectively. The average mass median aerodynamic diameter was estimated to be 2.32 µm based on graphic analysis of the particle size distribution as measured with a 1 ACFM Andersen Ambient Particle Sizing Sampler with an avergae geometric standard deviation of 1.98.
See Table 3.

Any other information on results incl. tables

Table 1. Summary of pre-test exposure trials




















Trial No.Trial Concentration (mg/L)Mass Median Aerodynamic Diameter (µm)
25.502.39
51.042.71

Table 2. Exposure conditions

































































Exposure Concentration (mg/L)5.071.05
Compressed Generator Air (Lpm)30.030.0
Compressed Mixing Air (Lpm)6.06.0
Total Air (Lpm)*36.036.0
Compressed Generator/Mixing air (psi)30/3030/30
T99 (min)²0.431.79
T99 (min) #0.863.58
Exposure Chamber Temp.(°C)2020-21
Exposure Chamber relative Humidity (%)2612-14
Ambient Room Temp. Range (%)19-2220-24
Total time of exposure (min)241244
Number of air changes per hour32277

*Total air = compressed generator air + compressed mixing air. ²Time for 90% equilibration of the chamber atmosphere. #Time for 99% equilibration of the chamber atmosphere.


Table 3. Characteristics of the achieved atmosphere






















































Exposure Concentration (mg/L)Sample No.Time of Sample (hours)Collection Time (min)Mean Mass Median Aerodynamic Diameter (µm)Geometric Standard Deviation
5.07 11.512.382.59
2312.182.38
Average 2.282.49
1.0511.522.461.95
 2322.182.01
Average 2.321.98

Table 4. Nominal Chamber Concentration:





























Atmosphere Concentration
Exposure concentration (mg/L)Total test substance used (g)Total Airflow (Lpm)Total time of exposure (min)Nominal concentration (mg/L)
5.07719.236.024182.90
1.05137.236.024415.62

Table 5. Body weights monitored during the study

















































































































































BODY WEIGHTS (GRAM)Mortality
ANIMALInitialDAY 1DAY 3DAY 7DAY 14DayWeighy (g)
1.05 MG/L
3307 (M)324322332338355--
3308 (M)335330350357377--
3309 (M)342334352357372--
3310 (F)220225237236241--
3311 (F)220221223215231--
3312 (F)220220226236240--
5.07 MG/L
3301 (M)255----0245
3302 (M)254----0246
3303 (M)263----0255
3304 (F)173----0171
3305 (F)168----0169
3306 (F)188----0185

Table 6. Mortality data summary:






























Exposure Concentration(mg/L)Number Dead/Number Tested
MalesFemalesCombined
5.073/33/36/6
1.050/30/330/6
LC50 (mg/L)Is between 1.05 mg/L and 5.07 mg/L (LC50 cut-off of 5 mg/L)

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Under the test conditions of this study, the inhalation LC50 (male/female) is between 1.05 mg/L and 5.07 mg/L (LC50 cut-off of 5 mg/L). The test material is classified in Category 4 (H332: Harmful if inhaled) according to Regulation (EC) No. 1272/2008 and the GHS as the LC50 value is comprised between 1 and 5 mg/L/4h.
Executive summary:

In an acute inhalation toxicity study (Acute Toxic Class Method) performed in accordance with GLP and OECD guideline 436, groups (3/sex) of Sprague Dawley rats were exposed by inhalation route for 4 hours to nose-only at a single exposure. After establishing the desired generation procedures during the pre-test trials,twelve healthy rats were selected for test and equally distributed into two exposure groups (3 males and 3 females per exposure): one exposure group with vapour concentation of 5.07 mg/L and second exposure group at 1.05 mg/L. Chamber concentration and particle size distributions of the test atmosphere were determined periodically during the exposure period.


Animals were then observed for mortality and clinical signs, gross toxicity and behavioral changes at least once daily for 14 days following exposure or until death occured. Bodyweights were recoeded prior to exposure (initial) and again on Days 1, 3, 7 and 14 (terminal) or after death. Necropsies were performed in all animals for macroscopical examination.


 


At the high dose level, the gravimetric chamber and nominal chamber concentrations were 5.07 mg/L and 82.90 mg/L, respectively. The average mass median aerodynamic diameter was estimated to be 2.28 µm based on graphic analysis of the particle size distribution as measured with a 1 ACFM Andersen Ambient Particle Sizing Sampler with an avergae geometric standard deviation of 2.49.
One female rat was found dead at chamber removal and the remaining five animals were euthanized for humane reasons following exposure to the test atmosphere. Prior to death, the euthanized animals were hypoactive and exhibited abnormal respiration, prone posture, and clonic convulsions. Gross necropsy of the decedents revealed discoloration of the lungs and/or distention of the intestines.


At the low dose level, the gravimetric chamber and nominal chamber concentrations were 1.05 mg/L and 15.62 mg/L, respectively. The average mass median aerodynamic diameter was estimated to be 2.32 µm based on graphic analysis of the particle size distribution as measured with a 1 ACFM Andersen Ambient Particle Sizing Sampler with an avergae geometric standard deviation of 1.98. All animals survived at the test item exposure and gained body weight during the study. Four rats were hypoactive and all animals exhibited abnormal respiration and/or ano-genital staining. However, all animals recovered by Day 3 and appeared active and healthy for the remainder of the 14-day observation period. No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.


Thus, the inhalation LC50 (male/female) is between 1.05 mg/L and 5.07 mg/L (LC50 cut-off of 5 mg/L). Therefore, the test material is classified in Category 4 (H332: Harmful if inhaled) according to Regulation (EC) No. 1272/2008 and the GHS as the LC50 value is comprised between 1 and 5 mg/L/4h.

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