2-ethoxy-1,3-dimethylcyclohexane

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 16 August 2016 and 06 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

guideline study Reliability 1 is assigned because the study conducted according to OECD TG 403 in co mpliance with GLP, without deviations that influence the quality of the results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed concentration procedure

Limit test:

no

Test material

Test material information

1

Reference substance name:

2-ethoxy-1,3-dimethylcyclohexane

EC Number:

944-610-3

Cas Number:

286472-48-8

Molecular formula:

C10H20O

IUPAC Name:

2-ethoxy-1,3-dimethylcyclohexane

Test material form:

liquid

Details on test material:

- Substance name as cited in test report: FRET 05-0293
- Phystical state: clear, colorless liquid
- Storage conditions: ambient temperature (15-25 °C), protected from light

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan™ : WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation:
- Weight at study initiation:
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:TEST ANIMALS
- Source:
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-12weeks
- Weight at study initiation: 200-350 g
- Fasting period before study: none
- Housing: in groups of up to five by sex in solid floor polypropylene cages with stainless steel lids, furnished with softwood flakes
- Diet (e.g. ad libitum): ad libitum (except during exposure)
- Water (e.g. ad libitum):ad libitum (except during exposure)
- Acclimation period: >5 days

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

IN-LIFE DATES: From: 16 August 2016 To: 06 December 2016

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

ca. 4.98 µm

Geometric standard deviation (GSD):

ca. 5.47

Remark on MMAD/GSD:

Particle Size Distribution
The particle size of the generated atmosphere inside the exposure chamber was determined three times during each exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). The device consisted of six impactor stages (10.4, 7.7, 4.1, 1.3, 0.9 and 0.56µm cut points) with stainless steel collection substrates and a backup glass fiber filter, housed in an aluminum sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump.
The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference.
The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 10.4, 7.7, 4.1, 1.3, 0.9 and 0.56 µm was calculated.
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.

Details on inhalation exposure:

Atmosphere Generation
The test item was aerosolized using a metal concentric jet nebulizer (Envigo CRS Limited, UK) located at the top of the exposure chamber. The nebulizer was connected to a plastic syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and to a metered compressed air supply.

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.

The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high). The concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump and air flow settings through the chamber. 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 diagram of the dynamic (continuous flow) system employed is shown in Figure 1.

Homogeneity of the test atmosphere within the chamber was not specifically determined during this study. Chambers of the same design (ADG Developments Ltd, Hitchin, Herts, UK) have been fully validated and shown to produce evenly distributed atmospheres in the animals’ breathing zone with a wide variety of test items (Green J D et al, 1984).

Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterization period test item input rates were varied in an attempt to achieve the required atmospheric conditions.

Sighting Exposure
During the characterization phase of the study, a group of two rats (one male, one female) were exposed to an atmosphere of the test item at a mean achieved atmosphere concentration of 2.34 mg/L for four hours. During exposure both animals exhibited decreased respiratory rate, the female animal also exhibited red/brown staining around the eye. On removal from the chamber the only significant observation noted in both animals was increased respiratory rate, the female animal also exhibited red/brown staining around the eye. One hour post-exposure, little or no change in the condition of the animals was apparent.
One day after the sighting exposure, both animals exhibited hunched posture and pilo-erection. Both animals recovered to appear normal on day 2 post-exposure.

Exposure Procedure
One day prior to the day of each 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.
Following an appropriate equilibration period one group of ten rats (five males and five females), was subjected to a single exposure of an atmosphere of the test item for a period of four hours. Based on the results of a sighting exposure, a target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 101 % of target and no deaths occurred, no further levels were required.

Exposure Chamber Temperature and Relative Humidity
The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every 30 minutes throughout each 4 Hour exposure period.

Exposure Chamber Oxygen Concentration
Oxygen levels within the exposure chamber were measured by an electronic oxygen analyzer (Servomex (UK) Ltd, Crowborough, East Sussex) located in a port in the animals breathing zone during the 4 Hour exposure period. The test atmospheres were generated to contain at least 19% oxygen.

Exposure Chamber Atmosphere Concentration
Prior to the inhalation phase of the study, the non-volatile component of the test item was determined by adding a small, known amount of test item to glass fiber filters and recording their weights. The filters were then dried in a desiccator at room temperature for approximately 24 hours and then weighed again. The difference in the two weights was taken as the volatile content of the test item and the non-volatile component was calculated as a percentage. The non-volatile component of the batch used during the study was found to be approximately 1.46 % (n=3). Due to the low levels of non-volatiles contained within this test item it was considered appropriate to use chemical analysis in order to determine test atmosphere concentrations throughout the exposure.
The test atmosphere was sampled nine times during each exposure period. The sampling procedure involved 2 liters of test atmosphere being drawn through a glass impinger containing Methanol (made up to 80mL). The samples were then submitted for chemical analysis.
The nominal chamber concentration was calculated by dividing the mass of test item used by the total volume of air passed through the chamber.
The nominal concentration was 127 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was relatively straight forward.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The analytical procedure had acceptable linearity and recoveries of test item in the impingers and was validated for use.

Duration of exposure:

ca. 4 h

Concentrations:

5.0 mg/L

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
Serial Observations
Clinical Signs
All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, 1 hour after termination of exposure and subsequently once daily for 14 days. Any evidence of overt toxicity was recorded at each observation.
Body Weight
Individual body weights were recorded on arrival, prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14.

Terminal Investigations
Necropsy
At the end of the 14 day observation period the surviving animals were killed by intravenous overdose of sodium pentobarbitone. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.

Results and discussion

Preliminary study:

During the characterization phase of the study, a group of two rats (one male, one female) were exposed to an atmosphere of the test item at a mean achieved atmosphere concentration of 2.34 mg/L for four hours. During exposure both animals exhibited decreased respiratory rate, the female animal also exhibited red/brown staining around the eye. On removal from the chamber the only significant observation noted in both animals was increased respiratory rate, the female animal also exhibited red/brown staining around the eye. One hour post-exposure, little or no change in the condition of the animals was apparent.
One day after the sighting exposure, both animals exhibited hunched posture and pilo-erection. Both animals recovered to appear normal on day 2 post-exposure

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.05 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

There were no unscheduled deaths during the study.

Clinical signs:

other: Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-Hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. These observations a

Body weight:

Four males and two female animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. Body weight gains were noted for all male animals during the remainder of the recovery period. In contrast, three female animals exhibited body weight losses from Days 1 to 3 post-exposure and four animals exhibited body weight losses or showed no body weight gains during the final week of the recovery period.

Gross pathology:

No macroscopic abnormalities were detected amongst animals at necropsy.

Other findings:

There were no other findings

Any other information on results incl. tables

The mean achieved atmosphere concentration was as follows:

Atmosphere Concentration
Mean Achieved (mg/L)	Standard Deviation	Nominal (mg/L)
5.05	0.17	6.43

The characteristics of the achieved atmosphere were as follows:

Mean Achieved Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)*	Geometric Standard Deviation*	Atmosphere estimated to be in the vapor phase (%)*
5.05	4.98	44.9	5.47	94.5

 *The results of the particle size distribution should be viewed with caution and are included only as a guide to the aerosol/vapor ratio of the generated atmosphere, as the volatility of a test item will be over-emphasized by the high air velocities and low pressures within a cascade impactor.

The mortality data were summarized as follows:

Mean Achieved Atmosphere Concentration (mg/L)	Deaths
	Male	Female	Total
5.05	0/5	0/5	0/10

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No deaths occurred in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.05 mg/L for 4 hours. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of the test item in the Wistar strain rat was greater than 5.05 mg/L

Executive summary:

A study was performed to assess the acute inhalation toxicity of the test item. The method used was designed to be compatible with that described in the OECD Guidelines for Testing of Chemicals (2009) No. 403 “Acute Inhalation Toxicity” and with Method B.2. Acute Inhalation Toxicity, 2014, of Commission Regulation (EC) No. 440/2008.

Methods

A group of ten RccHanTM : WIST strain rats (five males and five females) was exposed to an aerosol atmosphere.  The animals were exposed for 4 hours using a nose only exposure system, followed by a fourteen day observation period.

Results

The mean achieved atmosphere concentration was as follows:

Atmosphere Concentration
Mean Achieved (mg/L)	Standard Deviation	Nominal (mg/L)
5.05	0.17	6.43

The characteristics of the achieved atmosphere were as follows:

Mean Achieved Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)*	Geometric Standard Deviation*	Atmosphere estimated to be in the vapor phase (%)*
5.05	4.98	44.9	5.47	94.5

 *The results of the particle size distribution should be viewed with caution and are included only as a guide to the aerosol/vapor ratio of the generated atmosphere, as the volatility of a test item will be over-emphasized by the high air velocities and low pressures within a cascade impactor.

The mortality data were summarized as follows:

Mean Achieved Atmosphere Concentration (mg/L)	Deaths
	Male	Female	Total
5.05	0/5	0/5	0/10

 

Clinical Observations.  Common abnormalities noted during the study included decreased respiratory rate, increased respiratory rate, ataxia, hunched posture, pilo-erection, red/brown staining around the snout and wet fur.  Animals recovered to appear normal on Day 3 post-exposure.

Body Weight.  Four males and two female animals exhibited body weight losses or showed no body weight gain on the first day post-exposure.  Body weight gains were noted for all male animals during the remainder of the recovery period. In contrast, three female animals exhibited body weight losses from Days 1 to 3 post-exposure and four animals exhibited body weight losses or showed no body weight gains during the final week of the recovery period.  

Necropsy.  No macroscopic abnormalities were detected amongst animals at necropsy.

Conclusion

No deaths occurred in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.05 mg/L for 4 hours.  It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of the test item in the Wistar strain rat was greater than 5.05 mg/L.