Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 03 November 2015 and 11 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 403 using a standard acute method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

EC Number:

943-623-1

Molecular formula:

C13 H24 O

IUPAC Name:

Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

Test material form:

other: Liquid

Details on test material:

Identification: IFF 215 (Floriane)
Physical state/Appearance: clear colorless liquid
Storage Conditions: approximately 4 °C, in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry
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 five 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 eight to twelve weeks old and within the weight range of 200g to 350g. The females were nulliparous and non-pregnant.

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 (Datesand Ltd., Cheshire, UK) and provided with environmental enrichment items: wooden chew blocks and cardboard “fun tunnels” (Datesand Ltd., Cheshire, UK). With the exception of the exposure period, free access to mains drinking water and food (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water, bedding, chew blocks and cardboard “fun tunnels” are routinely analyzed and are considered not to contain any contaminants that could 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 25 °C 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 twelve hours continuous light (06:00 to 18:00) and twelve hours darkness. The animals were retained in this accommodation at all times except during the exposure period.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

Inhalation Exposure
Atmosphere Generation
The test item was aerosolized using a glass concentric jet nebulizer (Radleys, Saffron Walden, Essex, 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.

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 day of exposure, 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 exposed to an atmosphere of the test item for a period of four hours. After the results of the sighting study were available a target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 107 % of target and only one death occurred, no further levels were required.


Sighting Exposure
During characterization, 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 2.43 mg/L for approximately four hours. No significant effects were noted for either animal.


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 thirty minutes throughout the four-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 four-hour exposure period. The test atmosphere was 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 0.73 % (n=2). 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 for all exposure groups.

The test atmosphere was sampled nine times during the exposure period. The sampling procedure involved 2 liters of test atmosphere being drawn through a glass impinger containing Hexane (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 595 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was moderately difficult.


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 (8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 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 (%) of aerosol less than 8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 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.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Mean achieved atmosphere concentration was 5.33 mg/L (0.14 standard deviation); nominal concentration 31.7 mg/L.

No. of animals per sex per dose:

Five per sex per dose

Control animals:

no

Details on study design:

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, one hour after termination of exposure and subsequently once daily for fourteen days. Any deaths or evidence of overt toxicity were recorded at each observation.


Body Weight
Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14 or at death.


Necropsy
At the end of the fourteen day observation period the surviving animals were killed by intravenous overdose of sodium pentobarbitone. All animals, including the one that was humanely killed during the course of the study 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

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.33 mg/L air (nominal)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: One animal died in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.33 mg/L for four hours

Mortality:

One male died during the study. There was no female mortality.

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:

All male animals and four females exhibited body weight losses on the first day post-exposure. With the exception of one female animal which exhibited a slight body weight loss from Days 3 to 7 post-exposure, body weight gains were noted for all surviving animals during the final week of the recovery period.

Gross pathology:

Dark patches on the lungs were noted amongst two animals that survived until the end of the recovery period at necropsy, no macroscopic abnormalities were detected amongst the other surviving seven animals.

Dark patches on the lungs were noted at necropsy in the animal that was humanely killed during the course of the study:

Due to the clinical observations noted and macroscopic abnormalities detected, the contributing factor to the death noted during the study may have been mainly attributable to systemic toxicity.

Any other information on results incl. tables

Exposure Chamber Concentration

The actual concentration of the test item wasdeterminedby gaschromatography (GC). The test atmosphereswere sampled after theoretical chamber equilibration and then at approximately half-hourly intervals during the exposure period. The mean values obtained were:

 

Atmosphere Concentration
Mean Achieved (mg/L)	Standard Deviation	Nominal (mg/L)
5.33	0.14	31.7

 

The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.

The theoretical chamber equilibration time (T₉₉) was 3 minutes[1](Silver, 1946).

 

Particle Size Distribution

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

 

Mean Achieved Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)	Geometric Standard Deviation
5.33	3.40	57.0	2.50

[1] = Test atmospheres were generated for a total of 8 minutes prior to animal insertion to ensure test item concentration was being achieved.

Mortality Data

The mortality data are summarized as follows:

 

Mean Achieved Atmosphere Concentration (mg/L)	Deaths
	Male	Female	Total
5.33	1/5	0/5	1/10

 

Exposure Chamber Atmosphere Concentrations

Duration of Exposure (minutes)	Volume of Air Sampled (L)	Chamber Flow Rate (L/min)	Atmosphere Concentration (mg/L)
10	2	60	5.14
30	2	60	5.15
60	2	60	5.34
90	2	60	5.23
120	2	60	5.43
150	2	60	5.46
180	2	60	5.41
210	2	60	5.27
230	2	60	5.52

Mean achieved atmosphere concentration (mg/L) = 5.33

Standard deviation = 0.14

 

Nominal concentration:

Test item used (g)	472
Air Flow (L/min)	60
Total Generation Time (mins)	248[1]
Nominal Concentration (mg/L)	31.7

[1] = Test atmospheres were generated for a total of 8 minutes prior to animal insertion to ensure test item concentration was being achieved.

Particle Size Distribution

Cascade Impactor Data

Impactor Stage Number	Cut Point (µm)	Amount Collected (mg) per Sample Number			Mean Amount Collected (mg)
		1	2	3
3	8.4	0.28	0.29	0.25	0.27
4	7.3	0.53	0.56	0.52	0.54
5	3.6	0.53	0.64	0.70	0.62
6	1.3	0.76	0.81	0.92	0.83
7	0.94	0.31	0.36	0.38	0.35
8	0.43	0.03	0.02	0.05	0.03
Back-up Filter	<0.43	0.07	0.08	0.09	0.08
Total Mean Amount of Test Item Collected					2.72

 

Calculation 

Cut Point (µm)	Log10 Cut Point	Mean Cumulative Amount Less Than Cut Point
		(mg)	(%)	Probit
8.4	0.924	2.45	90.1	6.29
7.3	0.863	1.91	70.2	5.53
3.6	0.556	1.29	47.4	4.94
1.3	0.114	0.46	16.9	4.04
0.94	-0.027	0.11	4.04	3.25
0.43	-0.367	0.08	2.94	3.11

 

Results 

Mean Mass Median Aerodynamic Diameter (MMAD) = 3.40µm

Geometric Standard Deviation (GSD) = 2.50

Predicted amount less than 4 µm = 57.0%

Mortality Data

MeanAchievedAtmosphere Concentration (mg/L)	Sex	Deaths During Exposure	Deaths Post Exposure (1 Hour)	Deaths During Day of Observation								Total Deaths
				1	2	3	4	5	6	7	8-14
5.33	Male	0	0	1[1]	0	0	0	0	0	0	0	1/10
	Female	0	0	0	0	0	0	0	0	0	0

[1]= Humanely killed as considered unlikely to survive

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

One animal died in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.33 mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of IFF 215 (Floriane), in the RccHanTM : WIST strain rat, was greater than 5.33 mg/L.

Executive summary:

The acute toxicity of the test substance, IFF 215 (Floriane), via the inhalation route was assessed according to OECD Test Guideline 403 using a standard acute method. The 4 hr LC50 was greater than 5.33 mg/L.