Reaction mass of Ethyl 3-(cyclohex-1-en-1-yl) propanoate and Ethyl 3-(cyclohex-2-en-1-yl) propanoate

Endpoint summary

Description of key information

 Acute inhalation toxicity: OECD TG 403: LD50 > 4.78 mg/L 

Key value for chemical safety assessment

Acute toxicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 20 December 2016 and 8 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

yes

Specific details on test material used for the study:

Identification: FRET 11-0078
Physical state/appearance: clear colorless liquid
Storage Conditions: stored cold at approximately 4 °C in the dark

Species:

rat

Strain:

other: RccHan™ : WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
Male and female RccHan™ : WIST strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. After an acclimatization period of at least 5 days the animals were given a number unique within the study by ear punching and a number written on a color coded cage card. At the start of the study the animals were approximately 8 to 12 weeks old and within the weight range of 200 g to 350 g. The females were nulliparous and non pregnant.

Animal Care and Husbandry
The animals were housed in groups of up to five by sex in solid floor polypropylene cages with stainless steel lids, furnished with softwood flakes. With the exception of the exposure period, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25ºC and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness. The animals were retained in this accommodation at all times except during the exposure period.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

3.11 µm

Geometric standard deviation (GSD):

1.85

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 glass 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. 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.
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 to achieve the required atmospheric conditions.

Exposure Procedure
One day 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.
Following an appropriate equilibration period a single group of ten rats (five males and five females), was subjected to a single exposure to the test item for a period of four hours. A target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 96% 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 the 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.

Particle Size Distribution
The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). This device consisted of six impactor stages (10.4, 7.7, 4.1, 1.3, 0.90 and 0.56 µm 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 (percentage) of aerosol less than 10.4, 7.7, 4.1, 1.3, 0.90 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 (percentage) of aerosol less than 4 µm (considered to be the inhalable fraction) was determined.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

A target concentration of 5.0 mg/L was used for the exposure. The mean achieved concentration was 4.78 mg/L.

No. of animals per sex per dose:

5 males and 5 females

Control animals:

no

Details on study design:

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
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.

Data Evaluation
Data evaluations included the relationship, if any, between the animals’ exposure to the test item and the incidence and severity of all abnormalities including behavioral and clinical observations, necropsy findings, body weight changes, mortality and any other toxicological effects.
Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) of the test item was made.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 4.78 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality was observed

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

Body weight:

All animals exhibited body weight loss or no gain in body weight 1 day post exposure. Animals exhibited body weight gain from Days 1 to 3 post exposure except for two female animals which exhibited body weight loss or no gain in body weight. Body weight gain was noted for all animals during the remainder of the recovery period.

Gross pathology:

With the exception of two instances of dark patches on the lungs or pale lungs, no macroscopic abnormalities were detected amongst animals at necropsy.

Exposure Chamber Concentration

The actual concentration of the test item was measured off-line by gas chromatography using an external standard technique. The test atmospherewas sampled after theoretical chamber equilibration and then at approximately thirty minute intervals during the exposure period. Samples were then submitted for analysis.

The mean values obtained were as follows:

Group Number	Atmosphere Concentration
	Mean Achieved (mg/L)	Standard Deviation	Nominal (mg/L)
1	4.78	0.42	17.28

The chamber flow rate was maintained at 50 L/min providing 100 air changes per hour.

The theoretical chamber equilibration time (T₉₉) was 5 minutes* (Silver, 1946).

Particle Size Distribution

The particle size analysis of the atmosphere drawn from the animals’ breathing zone was as follows:

Group Number	Mean Achieved Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)	Geometric Standard Deviation
1	4.78	3.11	66.0	1.85

* = The test atmosphere was generated for a total of 19 minutes prior to animal insertion to ensure the target test item concentration was being achieved.

Interpretation of results:

GHS criteria not met

Conclusions:

The acute inhalation toxicity test showed an LD50 of > 4.78 mg/kg bw

Executive summary:

FRET 11-0078 was assessed for acute toxicity via inhalation using testing guideline OECD 403. In this study, 10 rats (5 males and 5 females) were exposed to an aerosol atmosphere of the test item at 4.78 mg/L. The rats showed no mortality and no clinical signs, changes in body weight or abnormalities at necropsy associated with toxicity of the test item. The inhalatory LD50 for the substance in male and female rats was determined to be > 4.78 mg/L.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

4 780 mg/m³ air

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Acute inhalation toxicity:

FRET 11-0078 was assessed for acute toxicity via inhalation using testing guideline OECD 403. In this study, 10 rats (5 males and 5 females) were exposed to an aerosol atmosphere of the test item at 4.78 mg/L. The rats showed no mortality and no clinical signs, changes in body weight or abnormalities at necropsy associated with toxicity of the test item. The inhalatory LD50 for the substance in male and female rats was determined to be > 4.78 mg/L.

Justification for selection of acute toxicity – oral endpoint
The result of this study is reliable and adequate for covering this endpoint.

Justification for classification or non-classification

According to the criteria outlined in Annex I of 67/548/EEC (DSD) and Annex VI of 1272/2008/EC (CLP), the substance does not have to be classified as acute toxic by the oral route.