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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-09-2013 to 23-01-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of (4RS,4aRS,8RS,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aSR)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one
EC Number:
939-400-3
Molecular formula:
C13H22O
IUPAC Name:
Reaction mass of (4RS,4aRS,8RS,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aSR)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark
- Other: clear colourless liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: Approximately the same age and body weight variation did not exceed +/- 20% of the sex mean. Females: 194-215 g and Males: 284 – 332 g
- Fasting period before study: None.
- Housing: Group housing of three animals per sex per cage in labelled Makrolon cages (type IV), containing sterilised sawdust bedding and paper cage enrichment.
- Diet (e.g. ad libitum): Certified diet from recognised supplier, provided ad libitum (except for exposure period period)
- Water (e.g. ad libitum): mains drinking water, ad libitum (except for exposure period period)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70 (actual: 32 – 40% ; was considered appropriate for this study)
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 06-09-2013 To: 30-09-2013

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 3.7 - <= 4.3 µm
Geometric standard deviation (GSD):
>= 1.9 - <= 2
Remark on MMAD/GSD:
MMAD/GSD relates to:
5 mg/L (nominal), 5.10 ± 0.11 mg/L (mean achieved concentration) dose : MMAD = 4.0 GSD = 2.0 and MMAD = 3.7 GSD = 2.0
1 mg/L (nominal), 1.03 ± 0.03 mg/L (mean achieved concentration) dose : MMAD = 3.9 GSD = 1.9 and MMAD = 4.3 GSD = 1.9
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: An aerosol was generated by nebulization of the test substance by means of a nebulizer and pressurized air. The primary aerosol was diluted with pressurized air before it entered the exposure chamber
- Exposure chamber volume: Not reported. Was consistent with Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983.
- Method of holding animals in test chamber: Restraining tubes.
- Source and rate of air: filtered air
- Method of conditioning air: Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the nebulizer.
- System of generating particulates/aerosols: nebulizer; the chamber mean total flow rate was maintained at 30 L/min (5 mg/L exposure level) and 33 L/min (1 mg/L exposure level).
- Method of particle size determination: Particle size was determined using a cascade impactor. The device consisted of eight impactors stages containing fiber glass filters.
- Treatment of exhaust air: From the exposure chamber the test atmosphere was passed through a filter before it was released to the exhaust of the fume hood.
- Temperature, humidity, in air chamber: The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter: 20.1-21.0°C, 32-40% humidity (this was considered appropriate for this study).

TEST ATMOSPHERE
- Brief description of analytical method used: Actual concentration was determined (n= 18 and 22 at 5 and 1 mg/L respectively) multiple times during the exposure period. Samples were drawn from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. Samples were drawn through a glass fibre filter. The collected amount of the test substance in the air sample was measured gravimetrically. Sample volumes were measured by means of a dry gas meter. The time-weighted mean concentration with the standard deviation was calculated. Full details of the analytical method are provided in the full study report.
- Samples taken from breathing zone: yes

VEHICLE
- Composition of vehicle (if applicable): Not applicable.
- Concentration of test material in vehicle (if applicable): Not applicable.
- Justification of choice of vehicle: Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: The particle size of the generated atmosphere inside the exposure chamber was determined two times during each exposure period using a cascade impactor. The particle size distribution for each group is reported in table 1.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The Mass Median Aerodynamic Diameter (MMAD) was determined and is reported for each group in table 1.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: In accordance with the OECD TG 436 guideline).
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
- 5.0 mg/L (1.89 mg/L nominal), 5.1 mg/L (mean achieved concentration) with a generation efficiency of 63%.
- 1.0 mg/L (8.0 mg/L nominal), 1.0 mg/L (mean achieved concentration) with a generation efficiency of 53%.
No. of animals per sex per dose:
5 per sex per dose. Full details are provided in table 2.
Control animals:
no
Details on study design:
- Duration of observation period following administration: 15 days
- Frequency of observations and weighing: Mortality, twice daily. Clinical signs three times during exposure and on day one at one and three hours and then once daily. Any evidence of mortality or overt toxicity was recorded at each observation. Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 4, 8 and 15 or after mortality.
- Necropsy of survivors performed: yes (and in the event of any mortalities)
- Other examinations performed: clinical signs, body weight, organ weights, and any other relevant toxicological effects were reported.
Statistics:
No statistical analysis was performed.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 1 - <= 5 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
At 5 mg/L : One male was humanely terminated on day 3 ; two females were humanely terminated on day 3.
At 1 mg/L : No mortality.
Clinical signs:
other: see "Any other information on results incl. tables"
Body weight:
At 5 mg/L : Reduced body weight gain and body weight loss was noted among survivors during the first week post exposure. All animals regained weight during the second week and/or gained weight over the course of the study.
At 1 mg/L : Reduced body weight gain and body weight loss was noted among survivors during the first week post exposure. All males/females regained weight during the second week and/or gained weight over the course of the study.
Gross pathology:
At 5 mg/L : Macroscopic post mortem examination of the two females humanely terminated revealed gelatinous contents in the stomach and/or duodenum. Macroscopic examination of the other females did not reveal any abnormalities. Pelvic dilation of the right kidney, as seen in one female exposed to 5 mg/L, is occasionally seen among rats of this age and strain and was therefore considered unrelated to treatment.
At 1 mg/L : No macroscopic abnormalities were noted.
Other findings:
- Other observations: The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity during necropsy.

Any other information on results incl. tables

Table 1.0 : Characteristics of the achieved atmosphere

Group Number

Nominal Concentration (mg/L)

Time-weighted mean actual concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (µm)

Geometric Standard Deviation

Comments

1 (1 mg/L)

1.89

1.03 ± 0.03

3.7 – 4.0

2.0

n=22 samples ; generation efficiency (ratio of actual and nominal concentration) of 53%

2 (5 mg/L)

8.0

5.10 ± 0.11

3.9 – 4.3

1.9

n=18 samples ; generation efficiency (ratio of actual and nominal concentration) of 63%

 

 

 

 

 

 

 

Table 2.0 : Mortality data

Group Number

Nominal Concentration (mg/L)

Time-weighted mean actual concentration (mg/L)

Mortalities

 

 

 

Female

Male

1 (1 mg/L)

1.89

1.03 ± 0.03

0/3

0/3

2 (5 mg/L)

8.0

5.10 ± 0.11

2/3

1/3

Clinical signs:

At 5 mg/L : slow breathing, during exposure. After exposure: lethargy, hunched posture, flat posture, uncoordinated movements, laboured respiration, ptosis, rales and/or hypothermia were observed. Surviving males/females had recovered by days 5 and 6.

At 1 mg/L : No clinical signs during or after exposure.

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the study, the inhalation 4h-LC50 (male/female) was considered to be > 1 and ≤ 5 mg/L and/or the LC50 cut off was considered to be 5 mg/L within the Crl:WI(Han) rat.
Executive summary:

The study was performed according to OECD TG 436 guideline in accordance with GLP to assess the acute inhalation toxicity of the test item. A single groups of six Wistar: Crl:WI(Han) strain rats (three males and three females) were exposed to an aerosol atmosphere of the test item. The groups were exposed for four hours using a nose only exposure system, followed by a fifteen day observation period. The time-weighted mean achieved atmosphere concentrations were as follows: 5.10 ± 0.11 mg/L based on a nominal concentration of 8.0 mg/L and/or 1.03 ± 0.03 mg/L based on a nominal concentration of 1.89 mg/L. The atmosphere generation efficiencies were 63% and 53%, respectively. The characteristics of the achieved atmosphere L were at 5 mg/: Mean Mass Median Diameter (particle size): > 3.7 μm and < 4.0 μm with geometric Standard Deviation 2.0 and 1 mg/L: Mean Mass Median Diameter (particle size): > 3.9 μm and < 4.3 μm with geometric Standard Deviation 1.9. At 5 mg/L, one male was humanely terminated on day 3 ; two females were humanely terminated on day 3. Slow breathing, was observed during exposure. After exposure lethargy, hunched posture, flat posture, uncoordinated movements, laboured respiration, ptosis, rales and/or hypothermia was observed. Surviving males/females had recovered by days 5 and 6. At 1 mg/L, there was no mortality and no significant clinical signs during or after exposure. Reduced body weight gain and body weight loss was noted among survivors during the first week post exposure. At all exposure levels. all animals regained weight during the second week and/or gained weight over the course of the study. At 5 mg/L, macroscopic post mortem examination of the two females humanely terminated revealed gelatinous contents in the stomach and/or duodenum. Macroscopic examination of the other females did not reveal any abnormalities. Pelvic dilation of the right kidney, as seen in one female exposed to 5 mg/L, is occasionally seen among rats of this age and strain and was therefore considered unrelated to treatment. At 1 mg/L, no macroscopic abnormalities were noted. Under the conditions of this study, the inhalation 4h-LC50 (male/female) was > 1 and ≤ 5 mg/L and the LC50 cut-off was considered to be 5 mg/L within the Wistar: Crl:WI(Han) rat.