4,8-Cyclododecadien-1-one

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-07-2014 to 09-10-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met with acceptable minor deviations (bodyweights of females were lower than 20% of the mean group body weights) but not considered to affect the purpose or validity of the study.

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

Due to availability, the following females were outside the weight range specified in the study plan (200-350g): 7f: 193g, 8f: 190g, 9f: 193g. This deviation is considered not to affect the purpose or validity of the study. Since were at the lower range.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Deviations:

yes

Remarks:

see above

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: March 2014 ; signature: May 2014

Test type:

traditional method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(3E,7E)-cyclododeca-3,7-dien-1-one; (4E,8Z)-cyclododeca-4,8-dien-1-one; (4Z,8E)-cyclododeca-4,8-dien-1-one

EC Number:

941-893-5

Cas Number:

15229-79-5

Molecular formula:

C12H18O

IUPAC Name:

(3E,7E)-cyclododeca-3,7-dien-1-one; (4E,8Z)-cyclododeca-4,8-dien-1-one; (4Z,8E)-cyclododeca-4,8-dien-1-one

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Approximately 4 °C, in the dark under nitrogen
- Other: colourless

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHanTM : WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 190-350 g; due to availability, the following females were outside the weight range specified in the study plan (200-350 g): 7f: 193g, 8f: 190g and 9f: 193g. With the exception of these females all body weights were within 20% of the mean weight for each sex, the deviation in relation to body weight for these three females was considered to not impact on the integrity of study. The applicant indicates: this is since the females were at the lower ranges of bodyweight.
- Fasting period before study: None.
- Housing: groups of up to 5 by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes and cage enrichment (wooden chew blocks and cardboard fun tunnels).
- 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): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): at least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks:

Oxygen concentration (%) was monitored during the study by an electronic oxygen analyzer. The test atmospheres were generated to contain at least 19% oxygen.

Mass median aerodynamic diameter (MMAD):

2.68 µm

Geometric standard deviation (GSD):

2.1

Remark on MMAD/GSD:

MMAD/GSD relates to: 35.1 mg/L (nominal), 4.53 mg/L (mean achieved concentration) dose

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The test item was aerosolized using a metal concentric jet nebulizer located at the top of the exposure chamber. The nebulizer was connected to a glass syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and to a metered compressed air supply.
- Exposure chamber volume: approximately 30 litres (dimensions: 28 cm diameter x 50 cm high)
- Method of holding animals in test chamber: Prior to the day of exposure each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. During the exposure period, each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.
- Source and rate of air: filtered air; chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.
- 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: metal concentric jet nebulizer; the concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump. The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.
- Method of particle size determination: Particle size was determined using a cascade impactor. The device consisted of six impactors stages. The mean amount for each stage was used to determine the cumulative amount below each cut-off point size to calculate the proportional (%) aerosol of defined size ranges. The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (%) of aerosol less than 4 μm (considered to be the inhalable fraction) was determined.
- Treatment of exhaust air: The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure. A schematic of the dynamic (continuous flow) system is presented in the full study report.
- Temperature, humidity, in air chamber: The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter: 20-22°C, 28-29% humidity.

TEST ATMOSPHERE
- Brief description of analytical method used: The test atmosphere was sampled nine times during the exposure period. The sampling procedure involved 2 to 10 litres (dependent on target concentration) of test atmosphere being drawn through drawn through a glass impinger containing Methanol (made up to 90mL). The sample was then submitted for chemical analysis by gas chromatography (GC). The test filter samples received were extracted with methanol to achieve the working concentration. Blank filters were accurately fortified with known amounts of test item and either analysed without further procedure or purged with nitrogen prior to analysis. A range of standard solutions were prepared in methanol from a stock solution of 1.041 mg/mL by serial dilution covering the concentration range 0.0 to 0.1562 mg/mL. Full details of the analytical method are provided in the full study report. The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. The regression coefficients calculated were found to be 0.999. The fortified samples of filters were found to have a recovery value of ± 10% of the fortification. The results indicate the accurate use of the test item and impingers during this study. Working solutions of the test item were shown to be sufficiently stable to complete the analysis without deterioration of the analyte. Full details of the analytical method are provided in the full study report.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: The particle size of the generated atmosphere inside the exposure chamber was determined three 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.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

35.1 mg/L (nominal), 4.53 mg/L (mean achieved concentration)

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: 14 days
- Frequency of observations and weighing: Clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for up to fourteen days. 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, 3, 7 and 14 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:

Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) was calculated using validated data analysis software which utilized Log-Normal (Probit) regression models in order to calculate the LC50 values (where possible). Alternatively, an estimate of the LC50 of the test item was made based on the available information.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 4.53 other: mg/l

Based on:

test mat.

Mortality:

1 female mortality occurred (humane sacrifice day 2) ; mortalities are reported in table 3.

Clinical signs:

other: Common abnormalities noted during the study included: During exposure one female (10f) animal exhibited decreased respiratory rate. On removal from the chamber all females exhibited body tremors and increased activity. All males/females also demonstrated

Body weight:

All animals exhibited body weight losses on the first day post-exposure. Body weight gains were noted in all surviving animals during the remainder of the recovery period.

Gross pathology:

Abnormally red lungs with dark patches were seen in 1 male (1m). 1 female exhibited pale liver and kidneys (10f). The female (8f) that was humanely sacrificed during the course of the study had dark liver and kidneys. No abnormalities were noted in any other males or females.

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

Additional comments regarding test atmosphere generation:

It is noted within the study that one sample (taken at 60 minutes of exposure) was lower than 20% of the mean achieved atmosphere concentration. As all other samples taken during the exposure were within the accepted 20 % of the mean achieved atmosphere concentration and only one animal mortality occurred this slight deviation to the test guideline was considered not to have affected the purpose or validity of the study. Rerunning the group was not considered appropriate within the study under the guideline. The results obtained (in terms of observations) would be considered to be similar on another group exposed at the same generation rates. It was considered that the test atmosphere consistency would not be able to be improved and the this was further confirmed by the large nominal concentration when compared to the mean achieved concentration.

 

Table 1. Characteristics of the achieved atmosphere:

Group Number	Mean Achieved Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)	Geometric Standard Deviation
1	4.53	2.68	70.7	2.10

 

Table 2. Mean achieved atmosphere concentrations:

Group Number	Mean Achieved Atmosphere Concentration (mg/L)	Standard Deviation	Nominal (mg/L)
1	4.53	0.48	35.1

 

Table 3. Mortality data summary:

Group Number	Mean Achieved Atmosphere Concentration (mg/L)	Mortalities
		Female	Male	Total
1	4.53	1/5	0/5	1/10

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the inhalation 4h-LC50 (male/female) was considered to be > 4.53 mg/L within the RCCHan WIST rat.

Executive summary:

The study was performed according to OECD TG 403 and EU Method B.2 in accordance with GLP to assess the acute inhalation toxicity of the test item. A single group of ten RccHanTM : WIST strain rats (five males and five 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 fourteen day observation period. The mean achieved atmosphere concentrations were as follows: 4.53 mg/L based on a nominal concentration of 35.1 mg/L. The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size) and Inhalable Fraction <4 μm were: 2.68 μm and 70.7% with geometric Standard Deviation 2.10. There was no male and one female mortalities in the 4.54 mg/L mean achieved atmosphere concentration. The single female mortality was a humane sacrifice. Observation changes noted during the study in the surviving animals included decreased respiratory rate, increased respiratory rate, ataxia, hunched posture, pilo-erection and wet fur. Occasional instances of body tremors, occasional body tremors and increased activity. Surviving animals recovered to appear normal from Days 7 to 8 post-exposure. Observations noted in the female that was humanely sacrificed during the study included decreased respiratory rate, ataxia, body tremors, occasional body tremors, increased activity, dehydration, lethargy, ptosis, splayed gait, hunched posture, pilo-erection and wet fur. All males and females exhibited body weight losses on the first day post-exposure. Body weight gains were noted in all survivors during the remainder of the recovery period. During necropsy abnormally red lungs with dark patches were seen in one male out of nine survivors. A single female survivor showed pale liver and kidney. No other macroscopic abnormalities were observed. In the single female mortality dark liver and dark kidney was seen at necropsy. A single mortality occurred in the group of five females and five males at the 4.53 mg/L mean achieved atmosphere concentration. Under the conditions of this study, the inhalation 4h-LC50 (male/female) was considered to be > 4.53 mg/L within the RCCHan WIST rat.