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Acute Toxicity: inhalation

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Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Principles of method if other than guideline:
The aim of this study was to determine the LC50 of the given test chemical in Wistar rats, after acute inhalation exposure for approximately 4 hrs and observation period of 14 days.
GLP compliance:
yes
Test type:
other: Acute Inhalation Toxicity
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol
EC Number:
266-100-3
EC Name:
4-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol
Cas Number:
66068-84-6
Molecular formula:
C16-H28-O
IUPAC Name:
4-{5,5,6-trimethylbicyclo[2.2.1]heptan-2-yl}cyclohexan-1-ol
Test material form:
liquid: viscous
Details on test material:
- IUPAC Name of the test chemical: 3-(2,3,3-trimethyl-6-bicyclo[2.2.1]heptanyl)cyclohexan-1-ol
- Common Name: Iso camphyl cyclohexanol
- Molecular Formula: C16H28O
- Molecular Weight: 236.396 g/mol
- SMILES Notation: OC1CCC([C@@H]2[C@@H]3C[C@@H](C(C)(C)[C@@H]3C)C2)CC1
- InChI: 1S/C16H28O/c1-10-14-8-12(16(10,2)3)9-15(14)11-4-6-13(17)7-5-11/h10-15,17H,4-9H2,1-3H3
- Substance Type: Organic
- Physical State: Colourless Viscous liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:In house breed.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 10 weeks at the time of exposure. Healthy young adult animals were used for the study.
- Weight at study initiation: Male: Maximum: 211 g; Minimum: 199 g
Female:Maximum: 191 g; Minimum: 180 g
- Housing:Group of three animals each were housed in polycarbonate cages.
- Bedding: All cages were provided with corn cobs.
- Room Sanitation:The experimental room floor and work tops were swept and mopped with disinfectant solution every day.
- Cages and water bottle:All the cages and water bottles were changed at least twice every week.
- Diet (e.g. ad libitum):All animals were provided conventional laboratory rodent diet, ad libitum.
- Water (e.g. ad libitum):Aqua guard filtered tap water was provided ad libitum via drinking bottles.
- Acclimation period: All animals were acclimatized to the test conditions for 7 days prior to exposure of the test item.
- Randomization: Animals were selected manually. No computer generated randomization program was used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):Minimum: 19.70°C; Maximum: 23.20°C
- Humidity (%):Minimum: 43.10 %; Maximum: 67.30 %
- Air changes (per hr):More than 12 changes per hour
- Photoperiod (hrs dark / hrs light):12 h light and 12 h dark

IN-LIFE DATES: From: March 20, 2015 To: April 13, 2015

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
other: Dimethyl sulfoxide (DMSO)
Mass median aerodynamic diameter (MMAD):
3.02 µm
Geometric standard deviation (GSD):
2.45
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the nose only inhalation chamber
- Exposure chamber volume: 22.52 liters
- Method of holding animals in test chamber: Each rat was accommodated in the portholes of inhalation chamber with the help of rat exposure tube. It was made up of transparent polycarbonate with adjustable unit and all rats were kept for four hour continuous exposure in inhalation chamber.
- Source and rate of air: Air compressor is used for supply of air to the inhalation unit. The applied air flow rate was 8 liter per minute
- Method of conditioning air: Air was passed into nebulizer at 30 psi pressure and 18 liter per minute air was drawn from the chamber to ensure slight negative pressure inside the chamber to prevent leakage of test item into the surrounding area
- System of generating particulates/aerosols: 70% formulated test item in Dimethyl sulfoxide (DMSO) was loaded into a 50 ml syringe and kept in infusion syringe pump. The infusion pump flow rate was 40 ml/hour. The test item was infused into nebulizer; aerosol was formed and distributed in the inhalation chamber.
- Method of particle size determination: The seven stage cascade impactor was used to determine the particle size.
- Treatment of exhaust air: The outgoing air from the chamber passes through conical flask containing 1% sodium hydroxide and moisture trap (cotton) by using the suction pump.
- Temperature, humidity, pressure in air chamber: The mean chamber temperature, relative humidity, oxygen and carbon dioxide concentration were 22.40°C, 54.03%, 21.08% and 0.09%, respectively; which were measured four times during 4 hour exposure period.

TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes, the breathing zone concentration and particle size were measured by using open face filter holder and seven stage cascade impactor four times during four hour exposure period.

VEHICLE
- Concentration of test material in vehicle (if applicable): 70% formulated test item in Dimethyl sulfoxide (DMSO)
- Lot/batch no. (if required): MMBB3970

TEST ATMOSPHERE (if not tabulated)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The MMAD was calculated according to the particle size at which the line across the 50% Mark whereas GSD was calculated according to the particle size at which the line across the 84.1% Mark divided by 50% Mark
Analytical verification of test atmosphere concentrations:
not specified
Duration of exposure:
4 h
Concentrations:
5.27 mg/l of air
No. of animals per sex per dose:
Three male and three female wistar rats
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical Observation - During inhalation exposure, individual animals were frequently observed at 1, 2, 3 and 4 hours and 30 minutes and one hour post exposure at day 0 (day of exposure). Subsequently, all animals were observed once a day during the 14 day observation period, after exposure.
Mortality - Animals were observed twice daily for mortality/morbidity during experimental period.
Body weight - All rats weighed on test days 0 (prior to application), 1, 3, 7 and 14 and found dead.
- Necropsy of survivors performed: yes, at the end of 14 day observation period, all surviving rats were euthanised by over dose of thiopentone sodium by intraperitoneal injection and subjected to gross pathology examination of external and internal observations. All animals showed no evidence of gross pathology hence, microscopic examination was not performed.
Statistics:
not specified

Results and discussion

Preliminary study:
not specified
Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.27 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
One mortality was observed at the breathing zone concentration of 5.27 mg/liter of air of test item during the 14 day observation period.
Clinical signs:
other: Clinical signs like lethargy and tremors were observed after exposure. Animal nos. 1, 2 and 4 were found normal from day 5 till terminal sacrifice whereas animal nos. 3 and 5 were found normal from day 6 till terminal sacrifice. Animal no. 6 was found dea
Body weight:
Decrease in mean body weight was observed on days 1 and 3 while increase in mean body weight was observed on days 7 and 14 in male rats whereas in female rats decrease in mean body weight was observed on day 1, 3, 7 and 14 when compared with day 0 mean body weight.
Gross pathology:
All the animals were subjected to gross pathological examination.
Other findings:
External Observation : External observation of found dead animal and terminally sacrificed animals did not show any pathological abnormality.
Internal Observation : Internal observation of all terminally sacrificed animals did not show any pathological abnormality.
In found dead animal following changes were observed in organs like: Lungs: Red discoloration was observed in Animal no. 6.

Any other information on results incl. tables

TABLE 1

Inhalation Chamber – Temperature, Relative Humidity, Oxygen, Carbon dioxide Concentration and Air Flow Rate During Exposure

Dose (mg/liter)

Time (Hours)

1

2

3

4

Mean

SD

5.27

Temperature (°C)

22.5

22.3

22.6

22.2

22.40

0.18

Relative Humidity (%)

53.9

54.2

53.5

54.5

54.03

0.43

Oxygen Concentration (%)

21.2

21.1

20.9

21.1

21.08

0.13

Carbon Dioxide Concentration (%)

0.05

0.09

0.13

0.08

0.09

0.03

In let Air Flow Rate              (liter per Minute)

8.00

8.00

8.00

8.00

8.00

0.00

Key :SD = StandardDeviation. 


TABLE 2

Concentration Measurement of Inhalation Chamber by Gravimetric Analysis

No.

Pre Weight (mg)

Post Weight (mg)

Difference in Weight (mg)

Air Suction Rate (LPM)

Time (minute)

Breathing Zone Concentration* (mg/l air)

Mean   ±

SD

1

26.44

36.98

10.54

2

1

5.27

5.27      ±             0.02

2

26.39

36.87

10.48

2

1

5.24

3

26.28

36.86

10.58

2

1

5.29

4

26.72

37.24

10.52

2

1

5.26

Keys :No. = Number, mg = Milligram,SD = Standard Deviation, LPM = Liter per Minute, l = liter

Differencein Weight (mg)

*BreathingZone Concentration                     =   ______________________________________

Air Suction Rate (liter/minute) X Time (minute)

 

 


TABLE 3

Nominal Concentration Details

 

Density of 70% formulated Test Item in Dimethyl Sulfoxide (DMSO)

 

No.

Amount of Sample (ml)

Weight of Sample (mg)

Mean±SD

1

1

1015.29

1015.71±0.41

2

1

1015.73

3

1

1016.11

 

Calculationof Nominal Concentration of Test Item

 

                                                                       Infusion Flow Rate (ml/ hour) X Density (mg/ml)

Nominal concentration (mg/l air)       =      _______________________________________       

                                                                                   Air Flow Rate per Hour (LPH)

 

Density (mg/ml)

Infusion Flow Rate (ml/hour)

Air flow rate (LPM))

Air flow Rate (LPH)

Nominal Concentration

(mg/liter)

1015.71

40

8

480

84.64

 

Keys :LPM = Liter per Minute, LPH = Liter per Hour,SD = Standard Deviation.


TABLE 4

Particle Size Distribution

Dose :5.27 mg/l of air

Stage

Effective Cutoff Diameter

Initial Weight (gm)

Final Weight (gm)

Difference in weight (gm)

% of Total Particles captured (by weight)

Cumulative (%)*

Sample 1

1

10.40

1657.35

1657.41

0.06

2.94

97.06

2

6.45

1660.63

1660.82

0.19

9.31

87.75

3

3.96

1647.35

1647.77

0.42

20.59

67.16

4

2.33

1638.78

1639.53

0.75

36.76

30.39

5

1.52

1661.30

1661.81

0.51

25.00

5.39

6

0.94

1659.64

1659.67

0.03

1.47

3.92

7

0.56

1653.56

1653.62

0.06

2.94

0.98

F

0.00

242.94

242.96

0.02

0.98

0.00

Sample 2

1

10.40

1657.35

1657.42

0.07

3.38

96.62

2

6.45

1660.64

1660.84

0.20

9.66

86.96

3

3.96

1647.35

1647.78

0.43

20.77

66.18

4

2.33

1638.79

1639.57

0.78

37.68

28.50

5

1.52

1661.31

1661.80

0.49

23.67

4.83

6

0.94

1659.64

1659.66

0.02

0.97

3.86

7

0.56

1653.56

1653.61

0.05

2.42

1.45

F

0.00

242.94

242.97

0.03

1.45

0.00

Keys: *= Percent of Particles smaller than corresponding effective cutoff diameter, F= filter
TABLE 4Continued...

Dose :5.27 mg/l of air

Stage

Effective Cutoff Diameter

Initial Weight (gm)

Final Weight (gm)

Difference in weight (gm)

% of Total Particles captured (by weight)

Cumulative (%)*

Sample 3

1

10.40

1657.35

1657.40

0.05

2.42

97.58

2

6.45

1660.63

1660.83

0.20

9.66

87.92

3

3.96

1647.35

1647.80

0.45

21.74

66.18

4

2.33

1638.79

1639.55

0.76

36.71

29.47

5

1.52

1661.31

1661.81

0.50

24.15

5.31

6

0.94

1659.64

1659.67

0.03

1.45

3.86

7

0.56

1653.56

1653.61

0.05

2.42

1.45

F

0.00

242.93

242.96

0.03

1.45

0.00

Sample 4

1

10.40

1657.35

1657.41

0.06

2.91

97.09

2

6.45

1660.63

1660.82

0.19

9.22

87.86

3

3.96

1647.35

1647.78

0.43

20.87

66.99

4

2.33

1638.78

1639.53

0.75

36.41

30.58

5

1.52

1661.31

1661.83

0.52

25.24

5.34

6

0.94

1659.64

1659.66

0.02

0.97

4.37

7

0.56

1653.56

1653.62

0.06

2.91

1.46

F

0.00

242.94

242.97

0.03

1.46

0.00

Keys: *= Percent of Particles smaller than corresponding effective cutoff diameter, F = Filter


TABLE 4Continued...

Summary of Particle Size Distribution

Collection Time :1 minute                                                  Air Flow Rate :2 liter per minute

 

Time

1

2

3

4

Mean±SD

MMAD

3.01

3.06

3.02

3.00

3.02 ± 0.03

GSD

2.45

2.47

2.44

2.45

2.45 ± 0.01

Keys:MMAD = Mass Median Aerodynamic Diameter,GSD = Geometric Standard Deviation, SD = Standard Deviation.


Applicant's summary and conclusion

Interpretation of results:
other: Not classified
Conclusions:
Under the conditions of this study, the median lethal concentration (LC50) of 70% of the given test chemical diluted in Dimethyl Sulfoxide (DMSO) in male and female Wistar rats was estimated as being greater than the breathing zone concentration of 5.27 mg/l air after four hour inhalation exposure and classified as “Not classified” as per CLP classification.
Executive summary:

Acute inhalation toxicity study of the given test chemical was conducted  as per OECD Guideline No. 403 in Wistar Rats.

Three male and three female healthy young adult rats were randomly selected and used for conducting acute inhalation toxicity limit study. Rats free from injuries were selected for the study. The study was carried out in dynamic nose only inhalation chamber. The dynamic inhalation chamber has three main parts namely inlet, exposure and outlet chambers. Total capacity of chamber is 22.52 liters. Each rat was restrained in a single transparent polycarbonate exposure tube with adjustable unit.

Continuous aerosol generation from as such (undiluted) test item was not possible hence test item was diluted in Dimethyl sulfoxide (DMSO) at the concentration of 70% (v/v) based on the solubility test. Formulated test item was loaded into a 50 ml syringe which was positioned to continuous infusion syringe pump at a flow rate of 40 ml/hour that was selected for main study based on pre test results. The rats were exposed to inhalation chamber exposure part. At a flow rate of 8 liter per minute, air was passed into nebulizer at 30 psi pressure and 18 liter per minute air was drawn from the chamber to ensure slight negative pressure inside the chamber to prevent leakage of test item into the surrounding area. Minimum 15 air changes per hour were maintained in inhalation chamber. The rats were exposed for continuous 4 hours after 20 minute equilibration period at the breathing zone concentration of 5.27 mg/liter air in Dimethyl Sulfoxide (DMSO). The mean chamber temperature, relative humidity, oxygen and carbon dioxide concentration were 22.40°C, 54.03%, 21.08% and 0.09%, respectively; which were measured four times during 4 hour exposure period. The mass median aerodynamic diameter of test item was 3.02 µm with geometric standard deviation of 2.45.

The animals were observed daily during the acclimatization period and mortality/morbidity and clinical signs were recorded. All animals were observed for clinical signs at 1, 2, 3 and 4 hours during exposure period and 30 minutes and one hour after exposure period on day 0. Mortality/Morbidity was recorded during 1, 2, 3 and 4 hours during exposure period and 30 minutes and one hour after exposure period on day 0 (in common with the clinical signs) and twice daily during days 1-14 (at least once on the day of sacrifice). Body weights were re­corded on day 0 (prior to exposure) and on days 1, 3, 7 and 14 and found dead. All animals were necropsied and examined macroscopically.

Clinical signs like lethargy and tremors were observed after exposure. Animal nos. 1, 2 and 4 were found normal from day 5 till terminal sacrifice whereas animal nos. 3 and 5 were found normal from day 6 till terminal sacrifice. Animal no. 6 was found dead on day 2 post exposure.

Decrease in mean body weight was observed on days 1 and 3 while increase in mean body weight was observed on days 7 and 14 in male rats whereas in female rats decrease in mean body weight was observed on day 1, 3, 7 and 14 when compared with day 0 mean body weight. ll the animals were subjected to gross pathological examination. External observation of found dead animal and terminally sacrificed animals did not show any pathological abnormality. Internal observation of all terminally sacrificed animals did not show any pathological abnormality. In found dead animal following changes were observed in organs like: Lungs: Red discoloration was observed in Animal no. 6.

Under the conditions of this study, the median lethal concentration (LC50) of 70% of the given test chemical diluted  in Dimethyl Sulfoxide (DMSO) in male and female Wistar rats was estimated as being greater than the breathing zone concentration of 5.27 mg/l air after four hour inhalation exposure and classified as “Not classified” as per CLP classification.