1-(5-ethyl-5-methylcyclohex-1-en-1-yl)pent-4-en-1-one

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25-03-2015 to 16-07-2015

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 not considered to impact the 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:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

three samples (total n=9) within 4 hour period were outside ±20% (two lower and one higher) the mean achieved concentration; not considered to impact the study; see "overall remarks, attachments" for further information

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: June 2015; signature: September 2015

Test type:

traditional method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-(5-ethyl-5-methylcyclohex-1-en-1-yl)pent-4-en-1-one

EC Number:

807-612-4

Cas Number:

1393645-32-3

Molecular formula:

C14H22O

IUPAC Name:

1-(5-ethyl-5-methylcyclohex-1-en-1-yl)pent-4-en-1-one

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Stored cold under nitrogen at approximately 4°C, in the dark
- Other: clear colourless

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: 8 - 12 weeks; females were nulliparous and non pregnant.
- Weight at study initiation: 200 - 350 g
- Fasting period before study: None.
- Housing: Housed in groups of up to five by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes provided with environmental enrichment items.
- Diet (e.g. ad libitum): Certified diet from recognised supplier, provided ad libitum (except for exposure period period)
- Water (e.g. ad libitum): ad libitum (except for exposure period period)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70%
- Air changes (per hr): > 15 air changes per hour
- Photoperiod: 12 h light / 12 h dark

IN-LIFE DATES: 25-03-2015 to 18-06-2015

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

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):

2.86 µm

Geometric standard deviation (GSD):

2.23

Remark on MMAD/GSD:

MMAD and GSD associated with the highest concentration level: 6.43 mg/L

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The test item was aerosolized using a glass 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: glass concentric jet nebulizer; the concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump.
- 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: 19 to 21 °C and 30 to 70% humidity. The relative humidity within the exposure chamber during the exposure was found to be lower (28 – 29 %) during the second exposure period than the range specified in the inhalation test guidelines (30 – 70 %). The decreased humidity may well have been due to the nature of the test item, the decreased humidity was therefore considered to be an accurate representation within the exposure chamber and is considered not to have affected the purpose or validity of the study.

TEST ATMOSPHERE
- Brief description of analytical method used: The test atmosphere was sampled nine times during each exposure period. The sampling procedure involved two liters or five liters of test atmosphere being drawn through a series of two glass impingers (dependent on concentration) containing Methanol (each made up to 80mL). The samples were then submitted for chemical analysis by gas chromatography (GC). The test impinger samples received were diluted with methanol to achieve the working concentration. The solvent (methanol) and a blank impinger (control) were analysed. Neither the solvent nor the blank impinger produced a signal that interfered with the signal due to the test item. A range of standard solutions were prepared in methanol from a stock solution of 1.030 mg/mL by serial dilution covering the concentration range 0 to 0.1545 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 0.9999. The fortified samples of impingers were found to have a recovery value of ± 10% of the fortification. the chromatographic run. In conclusion, 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
- 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:

Following an appropriate equilibration period three groups were subjected to a single exposure to the test item for a period of four hours. Based on the expected toxicity of the test item, a target concentration of 2.0 mg/L was used for the first exposure. Further concentrations were selected after consideration of the results of the previous exposure. Full details are provided in table 2.

No. of animals per sex per dose:

5 per sex per dose

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, bodyweight, 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. LC50 values and 95% confidence limits were calculated for males and females separately. Where appropriate.

Results and discussion

Effect levels

Effect levels 1

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 6.43 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: Effect levels based on analytically confirmed: mean achieved concentrations for each exposure/sex groups

Effect levels 2

Sex:

male

Dose descriptor:

LC50

Effect level:

> 6.43 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: Effect levels based on analytically confirmed: mean achieved concentrations for each exposure/sex groups

Effect levels 3

Sex:

female

Dose descriptor:

LC50

Effect level:

> 6.43 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: Effect levels based on analytically confirmed: mean achieved concentrations for each exposure/sex groups

Mortality:

There were no mortalities during the study as reported in table 3.

Clinical signs:

other: Common abnormalities noted during the study included increased respiratory rate, or decreased respiratory rate, noisy respiration, hunched posture, pilo-erection and wet fur. One female also demonstrated sneezing. All organisms in group 2 (6.43 mg/L mean

Body weight:

In group 2, 6.43 mg/L: All males and females exhibited body weight losses on Day 1 post-exposure. Body weight gains were noted in all males/females during the remainder of the recovery period.
In group 1, 2.14 mg/L: All males and four female animals exhibited body weight losses on Day 1 post-exposure. All male animals exhibited body weight gains during the remainder of the recovery period. One female animal showed no body weight gain from Days 1 to 3 post-exposure, however, body weight gains were noted in all females during the remainder of the recovery period.

All males/females were gaining bodyweight at the end of the recovery period.

Gross pathology:

In group 2, 6.43 mg/L: Abnormally red lungs or dark patches on the lungs were noted in two males (out of five) and two females (out of five) at necropsy.
In group 1, 2.14 mg/L: Dark patches on the lungs were noted in two males (out of five) and one female (out of five) at necropsy.

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. 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	2.14	2.41	77.7	1.96
2	6.43	2.86	66.3	2.23

 

Table 2. Mean achieved atmosphere concentrations:

Group Number	Atmosphere Concentration
	Mean Achieved (mg/L)	Standard Deviation	Nominal (mg/L)
1	2.14	0.04	6.87
2	6.43	1.92	21.7

 

Table 3. Mortality data summary:

Group Number	Mean Achieved Atmosphere Concentration (mg/L)	Deaths
		Male	Female	Total
1	2.14	0/5	0/5	0/10
2	6.43	0/5	0/5	0/10

The target concentrations were group 1: 2.00 mg/L and group 2: 5.00 mg/L, respectively

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the inhalation LC50 (male/female) was: > 6.43 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. Two groups 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: Group 1: 2.14 mg/L and Group 2: 6.43 mg/L. The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size) and Inhalable Fraction <4 μm: Group 1: 2.41 μm and 77.7% and Group 2: 2.86 μm and 66.3%. The Geometric Standard Deviation was Group 1: 1.96 and Group 2: 2.23, respectively. There were no male or female mortalities in Group 1 or Group 2. In Group 1, all males and four female animals exhibited body weight losses on Day 1 post-exposure. All male animals exhibited body weight gains during the remainder of the recovery period. One female animal showed no body weight gain from Days 1 to 3 post-exposure, however, body weight gains were noted in all females during the remainder of the recovery period.. Within Group 2, all males and females exhibited body weight losses on Day 1 post-exposure. Body weight gains were noted in all males/females during the remainder of the recovery period. In Group 1, One day after exposure, all animals exhibited increased respiratory rate, hunched posture and pilo-erection and one female animal also exhibited sneezing, which persisted for three days. All animals subsequently appeared normal on Day 5 post-exposure. In Group 2, on removal from the chamber all animals exhibited noisy respiration, there were frequent instances of increased respiratory rate and isolated occurrences of decreased respiratory rate. One hour post-exposure, all animals exhibited noisy respiration, all male animals exhibited increased respiratory rate and all females exhibited decreased respiratory rate. One day after exposure, all animals exhibited hunched posture and pilo-erection and there were frequent instances of increased respiratory rate. One male animal also exhibited decreased respiratory rate and noisy respiration. All males and females appeared normal from days 5 to 6 post-exposure. During necropsy in Group 1, dark patches on the lungs were noted in two males (out of five) and one female (out of five). In Group 2, abnormally red lungs or dark patches on the lungs were noted in two males (out of five) and two females (out of five) at necropsy. Under the conditions of this study, the inhalation LC50 (male/female) was > 6.43 mg/L within the RCCHan WIST rat.